The Veterinary Journal 197 (2013) 128–142

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The Veterinary Journal

journal homepage: www.elsevier.com/locate/tvjl

Review

Early embryonic development, assisted reproductive technologies,

and pluripotent stem cell biology in domestic mammals
V. Hall a,1, K. Hinrichs b,1, G. Lazzari c,1, D.H. Betts d,1, P. Hyttel a,⇑
a
Department of Veterinary Clinical and Animal Sciences, University of Copenhagen, Denmark
b
Department of Veterinary Physiology and Pharmacology, Texas A&M University, College Station, TX, USA
c
Avantea, Laboratory of Reproductive Technologies, Cremona, Italy
d
Department of Physiology and Pharmacology, Schulich School of Medicine and Dentistry, University of Western Ontario, London, ON, Canada

a r t i c l e i n f o a b s t r a c t

Article history: Over many decades assisted reproductive technologies, including artificial insemination, embryo transfer,
Accepted 4 May 2013 in vitro production (IVP) of embryos, cloning by somatic cell nuclear transfer (SCNT), and stem cell cul-
ture, have been developed with the aim of refining breeding strategies for improved production and
health in animal husbandry. More recently, biomedical applications of these technologies, in particular,
Keywords: SCNT and stem cell culture, have been pursued in domestic mammals in order to create models for
In vitro fertilization human disease and therapy. The following review focuses on presenting important aspects of pre-
Cloning
implantation development in cattle, pigs, horses, and dogs. Biological aspects and impact of assisted
Stem cells
Embryos
reproductive technologies including IVP, SCNT, and culture of pluripotent stem cells are also addressed.
Oocytes Ó 2013 Elsevier Ltd. All rights reserved.

Introduction developmental and stem cell biology in the larger domestic mam-
mals, and thus, the understanding of molecular and cellular aspects
Over the past decade, the landscape for veterinary research in of initial embryology and phenomena such as pluripotency and cell
embryo technology and stem cell biology has reshaped dramati- differentiation in these species is exponentially evolving.
cally. The initial focus of embryo technology in the domestic ani- The understanding of pre-implantation embryonic develop-
mals was to optimize breeding for improvement of production ment is a key to optimizing the use of domestic animals as models
and health. In some countries, such as Brazil and Argentina, em- for human disease, e.g. via refinement by genetic modification and
bryo technologies have found extended practical application, and establishment of different stem cell tools, as well as for optimizing
large numbers of bovine embryos are produced in vitro and trans- the use of embryo technologies for breeding and production. The
ferred to recipients in these regions. In most parts of the world, present review is an attempt to analyse current knowledge of the
however, the breeding-related use of such technologies is quanti- molecular aspects of pre-implantation development in pigs, cattle,
tatively limited. Investigations on pluripotent embryonic stem horses, and dogs as well as to discuss the significance of this
cells (ESCs) were initiated more than two decades ago with the knowledge for the practical refinement and utilization of in vitro
aim of using the technology for the production of genetically-mod- production of embryos, cloning by somatic cell nuclear transfer,
ified domestic animals. However, these initial efforts to establish and pluripotent stem cell culture.
ESCs in the domestic species were soon abandoned, due to the dis-
couraging results and, more importantly, to the ground-breaking The anatomy of pre-implantation embryonic development in
discovery that cultured embryonic or even somatic cells could be domestic mammals
reprogrammed into totipotency by the egg cytoplasm, allowing
for generation of genetically-modified animals by nuclear transfer. Proper maturation of the oocyte to metaphase II is a prerequi-
Recently, however, renewed focus on domestic animal embryo site for fertilization and pre-implantation development. In the
technology and stem cell biology has emerged, due to the need sow, cow, and mare maturation occurs in the pre-ovulatory follicle
for improved biomedical models for human diseases. This develop- within approximately the last 42, 24, and 36 h before ovulation,
ment has sparked in-depth research into fundamental aspects of respectively. Interestingly, in the dog the oocyte is ovulated with
an intact germinal vesicle and completes maturation in the oviduct
⇑ Corresponding author. Tel.: +45 35332541. over a 2–4 day period.
E-mail address: poh@sund.ku.dk (P. Hyttel). Upon fertilization, major embryonic genome activation, which
1
These authors contributed equally to this work. occurs at the 4-cell stage in pigs and around the 8-cell stage in cat-

1090-0233/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tvjl.2013.05.026
 V. Hall et al. / The Veterinary Journal 197 (2013) 128–142 129

Fig. 1. Initial development of the bovine embryo. A: Zygote; B: 2-cell embryo; C: 4-cell embryo; D: Early morula; E: Compact morula; F: Blastocyst; G: Expanded blastocyst;
H: Blastocyst in the process of hatching from the zona pellucida; I: Ovoid blastocyst with embryonic disc; J: Elongated blastocyst; K: Embryonic disc in the process of
gastrulation. 1: Inner cell mass; 2: Trophoblast; 3: Epiblast; 4: Hypoblast; 5: Embryonic disc; 6: Amniotic folds; 7: Ectoderm; 8: Mesoderm; 9: Endoderm (from Hyttel et al.,
2009).
130 V. Hall et al. / The Veterinary Journal 197 (2013) 128–142

tle, horses, and dogs, paves the way for the first lineage segregation
into trophoblast and inner cell mass (ICM; Fig. 1). Around the time
of hatching from the zona pellucida, the next lineage segregation of
the ICM results in the formation of the pluripotent epiblast and the
hypoblast, which develops into a flat layer epithelium that gradu-
ally covers the inside of the epiblast and trophoblast. The hypo-
blast is referred to as the primitive endoderm in the mouse; a
structure which should not be confused with the definitive endo-
derm (see later). Along with this process, the trophoblast trans-
forms from being a flattened cell layer to a more cuboidal cell
architecture. Subsequently, the polar trophoblast, covering the epi-
blast, referred to as Rauber’s layer, becomes increasingly thin, and,
finally, the epiblast penetrates the trophoblast and establishes the
embryonic disc, which thus becomes part of the outer lining of the
conceptus exposed to the uterine environment.
The following process of gastrulation results in formation of the
mesoderm, endoderm (i.e. the definitive endoderm) and ectoderm
(Fig. 1): first, the primitive streak develops through cell migration,
and cells continuously entering the streak undergo epithelial–mes-
enchymal transition and ingress to form mesoderm that spreads
between the trophoblast/epiblast and hypoblast, and endoderm,
which becomes inserted into the sub-epiblast portion of the hypo-
blast (i.e. definitive endoderm which becomes inserted into prim-
itive endoderm). The fate of the ingressing cells depend on their
site of ingression: those ingressing through the anterior streak
and primitive node become prechordal plate mesoderm, notochord
and endoderm; cells ingressing through ‘mid’ streak become par-
axial mesoderm, and cells ingressing through the posterior streak
become extra-embryonic and lateral plate mesoderm (Mikawa
et al., 2004). Fig. 2. In vitro production (IVP) of embryos in cattle. Immature oocytes are
From the time when the trophoblast gradually becomes lined aspirated from live animals by ultrasound-guided ovum pick up (1) or from abattoir
by extra-embryonic mesoderm on the inside, and becomes contin- ovaries (10 ). Immature oocytes at prophase I are submitted to in vitro maturation
uous with the ectoderm, through the transformation of the non- (IVM, 2) resulting in progression of meiosis to metaphase II, in vitro fertilization
(IVF, 3) resulting in pronucleus formation and initial cleavages, and in vitro culture
ingressing epiblast into ectoderm, the term trophectoderm is ap-
(IVC, 4) to the morula or blastocyst stage, at which time they can be transferred to
plied to this cell compartment. The term trophoblast will, again, recipients (from Hyttel et al., 2009).
be used for those cells of the trophectoderm, which engage in
forming the placenta. At the time of gastrulation, chorio-amniotic
folds consisting of trophectoderm with an inner lining of extra- In vitro production (IVP) of embryos
embryonic mesoderm develop into the amnion. A marked elonga-
tion of the conceptus occurs in pigs (to about a meter) and cattle at The birth of the first IVF calf derived from in vivo matured oo-
the time of gastrulation, whereas this phenomenon is not observed cytes in 1982 (Brackett et al., 1982) and the discovery of heparin
in horses and dogs. as capacitating agent for bull sperm 1986 (Parrish et al., 1986)
were the two key events that started an era of intense research ef-
forts for developing efficient bovine in vitro embryo production
Cattle (Bos primigenius taurus and Bos primigenius indicus) (IVP) procedures including in vitro maturation (IVM) of the oocyte
to the metaphase II, in vitro fertilization (IVF), and subsequent
Molecular regulation of pre-implantation development in vitro culture (IVC) of embryos to the blastocyst stage (Fig. 3).
The initial lack of knowledge on embryo requirements was by-
In cattle the major activation of embryonic genome occurs at passed by temporary in vivo culture in the surrogate sheep oviduct
the 8-cell stage (Fig. 2; Kues et al., 2008) accompanied by changes (Galli et al., 2003a, 2003b). At the same time co-culture with ovi-
in chromatin structure such as acetylation of core histones (Memili duct cells, Vero cells, BRL cells, granulosa cells was developed fol-
and First, 1999). At the 32–64-cell stage compaction occurs fol- lowed by cell-free methods based on synthetic oviductal fluid
lowed by blastulation at day 7–8, and hatching occurs at day 8–9 formulations (SOF; Gardner et al., 1994). While over 30% blastocyst
followed by elongation until implantation starts on day 20–21. formation could be achieved in most culture systems, it soon be-
Around hatching, the ICM cells differentiate into an inner layer came obvious that quantity did not always match quality (Loner-
facing the blastocyst cavity, the hypoblast, while the remaining gan et al., 2006) and that serum supplementation was
cells form the epiblast (Vejlsted et al., 2006). The polar trophoblast, detrimental to embryo/fetal development as the main causal factor
Rauber’s layer, soon degenerates and at the same time gene of the so-called large offspring syndrome (LOS), characterised by
expression changes of key pluripotency transcription factors abnormally advanced embryonic and fetal growth, altered gene
POU5F1, SOX2, and NANOG take place (Khan et al., 2012). Contrary expression patterns, and high perinatal losses (Young et al.,
to the mouse, this core triad is not confined to the ICM in the early 1998; Lazzari et al., 2002). A large field study demonstrated that
blastocyst, but it is also expressed by the trophoblast together with the incidence of LOS was greatly reduced by in vitro culture in
trophoblast-specific genes CDX2, HAND1, ETS2, and IFN-tau. After cell-free and serum-free SOF media (van Wagtendonk-de Leeuw
formation of the epiblast and hypoblast, however, POU5F1, SOX2, et al., 2000). At present the application of IVP combined with ovum
and NANOG expression becomes restricted to the epiblast (Degrelle pick up (OPU) from valuable donors is increasing due to developing
et al., 2005; Vejlsted et al., 2006). breeding strategies based on genomics selection using SNP (single
 V. Hall et al. / The Veterinary Journal 197 (2013) 128–142 131

(Wilmut et al., 1997), the first mammal to be cloned from a nucleus

of a somatic cell. Following this event several reports on cattle
cloned by SCNT emerged (Vignon et al., 1998; Galli et al., 1999).
While cloning originally was applied for enhancement of breeding
efficiency, including the rescue of a bovine breed close to extinc-
tion (Wells et al., 1998), it soon became a tool for obtaining genet-
ically modified calves (Cibelli et al., 1998a).
The SCNT technology involves the enucleation of a mature oo-
cyte creating a chromosome-free cytoplast, which is typically elec-
trofused with a diploid somatic cell (Fig. 4). This reconstructed
embryo is subsequently activated and embarks on embryonic
development. The subsequent steps that constitute the technology
have been refined with the contribution of dozens of laboratories
worldwide over several years. Mostly, enucleated metaphase II oo-
cytes (Campbell et al., 1996; Oback and Wells, 2003) are used as
recipient ooplasm, but also enucleated zygotes have been proven
suitable to reprogram the transferred nucleus in a more physiolog-
ical manner as compared to chemical activation of MII cytoplasts
(Schurmann et al., 2006).
Quiescent G0 is the preferred cell cycle stage of the donor so-
matic cells, but also cycling cells (Cibelli et al., 1998a) and blood
leukocytes (Galli et al., 1999) have been used as nuclear donors.
Technical modifications such as the zona-free manipulation have
improved the efficiency of enucleation and fusion (Oback et al.,
2003) in several species beside cattle (Lagutina et al., 2007)
although development to term is equal to conventional zona-en-
closed methods. SCNT is characterised by high pregnancy losses
occurring throughout gestation. A comparative embryo transfer
study between IVP embryos and cloned embryos derived from
embryonic, fetal, and adult cells provided evidence that while the
initial pregnancy rate at 21 days is similar (from 55.6% to 62.7%),
significant differences are evident at 70 days (49% vs. 37.3% vs.
22.5% vs. 14.3% for IVP, embryos and embryonic, fetal, and adult
cell clones, respectively) and at calving (49% vs. 34.3% vs. 15% vs.
6.8%; Heyman et al., 2002a). For fetal and adult somatic cell cloning
the efficiency is also influenced, for yet unclarified mechanisms, by
the specific cell line used as source of nuclei (Powell et al., 2004).
Several studies have demonstrated that SCNT embryos present
an altered gene expression (Smith et al., 2005) and epigenetic sta-
tus (Dean et al., 2001), compared with IVP embryos, and the high
rate of pregnancy loss has been clinically associated with hydrops
and cotyledonary hyperplasia (Heyman et al., 2002b; Everts et al.,
2008). These problems are, however, not observed in the offspring
of clones, which are normal (Heyman et al., 2004). Because of their
poor survival to term, cloned cattle have been subjected to intense
studies to demonstrate that the composition of milk and meat from
these animals is not different from controls (Heyman et al., 2007)
and products from cloned animals or their progeny do not pose
any health risk to the consumer.
Fig. 3. Bovine in vitro production of embryos. Bovine immature oocyte (A), 8-cell
embryo (B), and expanded blastocyst (C). 1: Cumulus cells; 2: Zona pellucida; 3: Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs)
Inner cell mass; 4: Blastocyst cavity. Scale bars: 50 lm (A, B, and C).
ESCs were isolated for the first time from the murine ICM and
cultured and characterized by Evans and Kaufman (1981) and Mar-
nucleotide polymorphism) chips, whereby thousands of genetic tin (1981). Recent evidence suggests that there are distinct states
markers can be screened even on a small embryo biopsy before of pluripotency (naïve and primed) that differ both morphologi-
embryo transfer, with enough precision to anticipate that the need cally and functionally (De Los Angeles et al., 2012). Naïve murine
for progeny testing will be dramatically reduced. ESCs (Evans and Kaufman, 1981; Martin, 1981) are derived from
the ICM or early epiblast cells, proliferate in culture as packed
Somatic cell nuclear transfer (SCNT) dome-like colonies, are maintained in the undifferentiated state
by LIF-JAK-STAT3 and BMP4 signalling, readily contribute to chi-
In 1986, Willadsen obtained the first cloned sheep using nuclei meric embryos, maintain two active X chromosomes (in female
of embryonic blastomeres for nuclear transfer (Willadsen, 1986). cells) and are relatively resistant to differentiation into primordial
The following year this result was reproduced in cattle (Prather germ cells (PGCs) and extra-embryonic lineages (Kuijk et al., 2011).
et al., 1987). These achievements sparked a period of intense re- In contrast, primed pluripotent stem cells have been derived
search in farm animals, which culminated with the birth of ‘Dolly’ from epiblasts of post-hatching murine blastocysts, are termed
132 V. Hall et al. / The Veterinary Journal 197 (2013) 128–142

Fig. 4. Cloning of sheep by somatic cell nuclear transfer (SCNT). Oocytes are collected from a donor, matured in vitro to metaphase II, and enucleated (usually by
micromanipulation) to remove the first polar body and the portion of the oocyte containing the metaphase plate. The resulting cytoplast is combined with a donor cell (e.g. a
cultured fibroblast cell) originating from the animal to be cloned. The cytoplast and donor cell are submitted to electrofusion resulting in production of reconstructed
embryos in which the oocyte cytoplasm is mixed with the donor cell cytoplasm and carries the donor cell nucleus. The reconstructed embryos are activated in order to initiate
embryonic development and, after culture for about 1 week to the blastocyst stage, can be transferred to recipients for development into cloned offspring (from Hyttel et al.,
2009).

epiblast stem cells (EpiSCs), are molecularly and epigenetically dif- All reported ESC-like bovine cells do not proliferate long-term,
ferent from murine ESCs (Brons et al., 2007; Tesar et al., 2007), except for a few cases (Mitalipova et al., 2001), and are not capable
have a more flattened colony morphology, depend on basic fibro- of contributing significantly to chimeras following morula aggrega-
blast growth factor (bFGF) or transforming growth factor alpha tion (Iwasaki et al., 2000). A recent study (Maruotti et al., 2012) has
(TGFa)/activin signalling for self-renewal, exhibit a limited ability described the application of human ESC and mouse EpiSC culture
to contribute to chimeras, have undergone X-chromosome inacti- protocols, based on bFGF and activin-nodal signalling, to post-
vation, and readily differentiate into PGC precursors in vitro (Brons hatching pre-implantation bovine blastocysts, but again undiffer-
et al., 2007). The optimal passaging procedure (single cells trypsin- entiated proliferation could not be maintained.
isation vs. disaggregation in clumps) and growth kinetics (14–16 h Induced pluripotent stem cells (iPSCs) were first produced in
doubling time in murine ESCs vs. 36 h in murine EpiSCs) also differ. the mouse in 2006, by inserting four transcription factors, includ-
Moreover, the expression of some pluripotency markers is differ- ing Pou5f1, Sox2, Klf4, and c-Myc into embryonic and adult cells,
ent: Pou5f1 (also known as Oct4), Nanog, and Sox2 are common, resulting in a reversion of these cells into a pluripotent state, sim-
but Klf4, Dppa3, and Zfp42 are specific for murine ESCs. Surpris- ilar to that observed in the ICM (Takahashi and Yamanaka, 2006).
ingly, ESCs derived from human blastocysts exhibit characteristics These pluripotent cells can now be created from a multitude of
more like those of murine EpiSCs than their murine ESC counter- different factors, cell backgrounds, and methods in many different
parts (Thomson et al., 1998). species (Hussein and Nagy, 2012). Derivation of bovine iPSCs was
Most of the published studies on attempting bovine ESC deriva- attempted (Huang et al., 2011) using transfection with a polycis-
tion have applied the original mouse protocols (Stice et al., 1996; tronic plasmid containing the complete bovine cDNAs for POU5F1,
Cibelli et al., 1998b; Mitalipova et al., 2001; Saito et al., 2003; Kee- SOX2, KLF4, and c-MYC, into bovine fibroblasts that were then cul-
fer et al., 2007) starting from 2-cell embryos (Mitalipova et al., tured in presence of specific signalling inhibitors successfully used
2001) up to day-12 hatched blastocysts (Gjorret and Maddox-Hyt- for mouse and rat ESC culture (Buehr et al., 2008; Ying et al.,
tel, 2005). Colony formation ranges from 14% to 70% in the differ- 2008). Reprogramming efficiency was 0.4% giving rise to non-
ent studies. Some authors report the morula as the most suitable proliferative dome-shaped colonies expressing markers of
stage (Stice et al., 1996) and others the day-8 blastocyst (Talbot pluripotency, including endogenous iPSC factors, CDH1, DPPA3,
et al., 1995). There is some controversy with respect to expression NANOG, SOCS3, ZFP42, telomerase, Tra-1-60/81, and SSEA-3/4,
of pluripotency markers: according to Saito et al. (2003), presump- but not SSEA-1.
tive bovine ESCs express alkaline phosphatase (AP), FUT4 (also In an another study a lentiviral expression vector (pLentilox 3.7)
known as SSEA1), STAT-3, and POU5F1, but are negative for SSEA4, for human POU5F1 and porcine SOX2, C-MYC, and KLF4 fused with
whereas other authors report that they are AP positive and stain EGFP was transduced into fetal fibroblasts obtaining a reprogram-
for SSEA4, POU5F1, TRA-1-81, and TRA-1-60 (Wang et al., 2005; ming efficiency of 0.0002–0.0007% in the presence of LIF and bFGF.
Munoz et al., 2008) and yet others consider AP staining negative The derived colonies resembled human ESCs rather than mouse
while FUT4, SSEA3, and SSEA4 positive (Stice et al., 1996; Cibelli ESCs, but the transgenes were only partially silenced, indicating
et al., 1998a, 1998b; Mitalipova et al., 2001). incomplete reprogramming (Cao et al., 2012).
 V. Hall et al. / The Veterinary Journal 197 (2013) 128–142 133

Pig (Sus scrofa domesticus)

Molecular regulation of pre-implantation development

The development of the porcine pre-implantation embryo is

dependent on a number of key cell signalling events. Initial embry-
onic cleavage is controlled primarily by innate maternal compo-
nents carried over from the oocyte in the form of RNA and
proteins. However, recent reports suggest that RNA may be intro-
duced via spermatozoa, which could contribute to initial develop-
ment. Early cleavage events may also be steered or enhanced by
external factors present in the oviduct such as oviduct-produced
proteins or luminal secreted factors (Buhi et al., 1997). A number
of key regulators have been found to act during the initial cleav-
ages including the cell cycle controlling phosphatase cdc25 family
(Kim et al., 1999; Anderson et al., 2001) and members of the Src
family kinase (SFK) family (Levi et al., 2010). The major embryonic
genome activation, which occurs at the 4-cell stage in the pig,
paves the way for more complex developmental progression
(Fig. 5; Jarrell et al., 1991).
This maternal to embryonic transition marks an important
event in development: largely, overcoming transcriptional silenc-
ing of the embryo. This event is partly attributable to chromatin
remodelling events. Chromatin remodelling genes, including Smar-
ca2, have been shown to be important in porcine embryonic cleav-
age (Magnani and Cabot, 2007). A number of histone
methyltransferases known to modulate H3K9 (which is associated
with transcriptional silencing and cleavage control) have been
found to be important in porcine embryonic cleavage (Park et al.,
2011). Low expression of the H3K27me3 methylase EZH2 and its
co-factors EED and SUZ12 at the 4-cell stage (Gao et al., 2010) sug-
gests a reversal of H3K27me3-dependent transcriptional silencing
occurs at this stage. Furthermore, histone H3K4me3 (implicated in
gene activation) is thought to play a particular role in the mater-
nal-to-embryonic transition in the pig (Gao et al., 2010).
The first differentiation event, occurring as the blastocyst forms,
appears to be also regulated by key genes. Similar to the mouse,
ELF5 is expressed primarily in the porcine trophoblast and plays
an important role in trophoblast specification (Gao et al., 2011b).
CDX2 is another key gene expressed in the porcine trophoblast
(Gao et al., 2011a). Interestingly, Eomes, which is another impor-
tant marker for lineage segregation in the mouse, is dependent
on expression of Cdx2 in the trophoblast (Ralston and Rossant,
2008), however, is only expressed in the porcine epiblast and not
in the trophoblast (Wu et al., 2010).
Another important gene for ICM specification is probably
POU5F1, which is expressed in these cells and becomes exclusively
localized in the epiblast during later development (Hall et al.,
2009; Gao et al., 2011b). It remains unclear whether the genes NA-
NOG and SOX2 play an important role in ICM specification in the
pig. These genes are expressed in the murine ICM, but are only ex-
pressed in the pig epiblast (Hall et al., 2009; Wolf et al., 2011). Data
on gene regulation during early development are more advanced in
the well-studied mouse, but there are some reports which show Fig. 5. Porcine oocyte and embryos derived in vivo. Porcine mature oocyte (A), 8-
that both similarities and differences exist between the mouse cell embryo (B), and blastocyst (C). 1: Zona pellucida; 2: First polar body; 3: Inner
and the pig. cell mass; 4: Blastocyst cavity. Scale bars: 50 lm (A, B, and C).

In vitro production (IVP) of embryos 50% in some laboratories (Mugnier et al., 2009). Despite these dif-
ficulties, IVP blastocyst development rates tend to vary from 30% to
The pig has been a particularly difficult species in which to ob- 50% from monospermically-fertilized oocytes in most laboratories
tain high rates of fertilization and subsequent blastocyst develop- (Gil et al., 2010). Problems with mitochondria migration during
ment in vitro. Problems in oocyte cytoplasmic maturation IVM have been postulated to be one potential reason for lack of
in vitro, high rates of polyspermy, and low embryonic development developmental competence (Sun et al., 2001).
rates are the major obstacles that still need to be overcome (Gil The addition of particular components, such as porcine follicu-
et al., 2010). The rate of polyspermy has been reported to be over lar fluid, into the IVM media has been shown to improve the qual-
134 V. Hall et al. / The Veterinary Journal 197 (2013) 128–142

ity of porcine IVM (Algriany et al., 2004), as has hormones at par- will aid cancer research and xenotransplantation, such as the re-
ticular stages of maturation and insulin–transferrin–selenium (Hu cently produced SCID pig (Suzuki et al., 2012). Thus the pig is fast
et al., 2011). Due to a refinement of techniques, IVM rates now vary paving the way for an alternative biomedical animal model for
from 75% to 85% (Gil et al., 2010). One reason for the high rate of varying diseases.
polyspermy seen in this species may relate to a delay in the zona
reaction, which under normal conditions establishes a prompt bar-
rier to the fertilization by supernumerary spermatozoa (Wang Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs)
et al., 1998). Reducing the time of exposure of oocytes to the sper-
matozoa has led to an increase in monospermic fertilization (Gil Production of bona fide porcine ESCs remains elusive, which is
et al., 2010). A synthetic attempt to harden the zona has also been likely due to inadequate culture conditions (Hall, 2013). Several re-
attempted using an amine-reactive cross linker, which resulted in a search groups have attempted to produce porcine ESCs, however
5-fold increase in monospermic fertilization and an increase in fer- these cells only undergo a limited number of cell passages and dif-
tilization rate (Coy et al., 2008). ferentiate spontaneously in culture (Hall, 2008). One research
Embryo culture (IVC) has been developed extensively in the pig, group has successfully been able to culture porcine ICM cells fol-
and two particularly successful media compositions are used widely lowing transduction with the pluripotency genes POU5F1 and
today, including NCSU23 and NCSU-37 (Petters and Wells, 1993). KLF4 (Telugu et al., 2011), but transgene-free porcine ESCs remain
Two independent studies have shown that removal of glucose dur- lacking.
ing the first 48–72 h can significantly improve blastocyst develop- In contrast, iPSCs have been produced in the pig by several dif-
ment (Abeydeera, 2002; Kikuchi et al., 2002). A chemically defined ferent groups (Fig. 6; Ezashi et al., 2012; Kues et al., 2012). These
media has also been developed (PZM5) which appears to be very cell lines demonstrate pluripotency and have been shown to con-
successful for porcine embryo culture (Yoshioka et al., 2008). tribute towards the formation of chimeras (West et al., 2010) and
The rate of live offspring resulting from IVP is generally rela- may even be transmitted through the germline, although this type
tively low in the pig compared to the number of transferred IVP of transmission was considered rare and combined with perinatal
embryos, and to date, successful generation of offspring depends death potentially due to epigenetic aberrations (West et al.,
on transfer of large numbers of blastocysts or earlier embryos, to 2011). However, unlike their mouse and human counterparts,
produce sufficient litter sizes. A live offspring rate of 11% to 16% these cell lines do not silence their inserted transgenes, either dur-
has been reported from transferred blastocysts when using chem- ing culture or during cell differentiation (Ezashi et al., 2012; Hall
ically defined culture media (Kikuchi et al., 2002). The need to et al., 2012). Furthermore, the cells are unable to maintain pluripo-
transfer relatively large numbers of embryos to achieve even a tency and self-renew when the transgenes are turned off (Wu et al.,
comparatively low litter size, as well as the lack of stable non-sur- 2009), indicating that the cells are neither stable in vitro, nor fully
gical procedures for embryo transfer remain significant obstacles reprogrammed.
towards the practical implementation of IVP. In contrast, artificial Problems with both ESCs and iPSCs therefore remain and fur-
insemination (AI) remains a mainstream method for ART in swine ther research is required in order to determine the exact underly-
and is used across Europe, the USA, and many other countries ing causes for the continued problems of these cells in culture.
worldwide (Day, 2000). Transcriptional profiling of the naïve pluripotent ICM or epiblast
may provide some clues as to whether any differences exist in
Somatic cell nuclear transfer (SCNT) these cells compared to ESCs and iPSCs from mouse and human.
Some initial studies have already shown that differences do exist,
Over the past decade, the efficiency of SCNT has significantly such as the absence of NANOG and SOX2 in the porcine ICM (Hall
improved due to refinements and simplifications of the cloning et al., 2009). Thus, in vitro cell tools have been developed in the pig,
technology (Vajta and Callesen, 2012). Development of a zona-free
methodology, based on removal of the zona pellucida of the oocyte,
combined with the so-called hand-made-cloning, where the oocyte
is enucleated by simple hand-held bisectioning, are aspects that
have helped to improve blastocyst development of SCNT-produced
embryos in the pig (Lagutina et al., 2007; Vajta and Callesen, 2012).
Pre-treatment of porcine fibroblasts using Xenopus egg extract has
also led to improved in vitro SCNT embryo development (Liu et al.,
2011, 2012). As for IVP, vast numbers of SCNT embryos are trans-
ferred to produce pregnancies and even relatively-low litter sizes.
A recent report has shown that the average rate of offspring
from porcine SCNT embryos using two different pig breeds and
two different methods was approximately 7% of transferred em-
bryos (Schmidt et al., 2010). Perinatal mortality and malformations
are unfortunately still major issues that are reported in this species
(Schmidt et al., 2010). This is considered to be caused by errors in
epigenetic and genetic reprogramming, and has been overcome in
some studies by performing an additional re-cloning step
(Fujimura et al., 2008; Cao et al., 2012). Despite these considerable
Fig. 6. Porcine induced pluripotent stem cell-like cells. Bright field image of a
setbacks, a staggering number of genetically-modified pigs, serving colony reveals small compact cells containing a large nuclear-to-cytoplasmic ratio
as potential human disease models, have been produced using with prominent single nucleoli (marked with arrow) and typical high lipid content
SCNT, including animals carrying gene modifications potentially (refractive yellow portions of colony). Colonies also express alkaline phosphatase
resulting in skin inflammation related to psoriasis (Staunstrup (inset image). Colonies are at passage 4 and were grown on mitomycin C-treated
mouse embryonic fibroblasts. Cell culture conditions used were KnockOut-DMEM
et al., 2012), Alzheimer’s disease (Kragh et al., 2009), cystic fibrosis containing 15% KnockOut Serum, 1 non-essential amino acids, 1 glutaMAX, 1
(Welsh et al., 2009; Klymiuk et al., 2012), and diabetes (Renner beta-mercaptoethanol, 1 penicillin–streptomycin and supplemented with MEK
et al., 2010). Transgenic pig models have also been developed that inhibitor PD0325901 and GSK3 inhibitor, CHIR99201. Scale bar: 100 lm.
 V. Hall et al. / The Veterinary Journal 197 (2013) 128–142 135

however, further refinement of culture conditions is warranted in via transvaginal oocyte aspiration (TVA; Brück et al., 1992; Colleoni
order to stabilize iPSC cells and enhance their reprogramming. et al., 2007; Jacobson et al., 2010). Mature oocytes may be collected
Such advances may also allow for the development of porcine ESCs by aspiration of the dominant pre-ovulatory follicle after gonado-
in the future. tropin stimulation, via TVA or puncture through the flank (Carne-
vale and Ginther, 1993; Hinrichs et al., 1998).
Immature oocytes may be held overnight in a modified M199 at
Horse (Equus ferus caballus)
room temperature before maturation, with no effect on develop-
ment (Choi et al., 2006). Maturation is performed effectively in
Molecular regulation of pre-implantation development
M199 with fetal bovine serum and FSH (Hinrichs et al., 2005; Choi
et al., 2007; Ribeiro et al., 2008). The optimum duration of matura-
The time of major embryonic genome activation in the horse
tion is 24–30 h and 30–36 h for oocytes initially having expanded
embryo appears to be around the 6-cell stage (Brinsko et al.,
and compact cumuli, respectively. The maturation rate of compact
1995; Grondahl and Hyttel, 1996). The equine blastocyst cavity
oocytes is lower than that for expanded oocytes (20% vs. 65%),
forms in a multicentric manner, resulting in a loose network of in-
but there is no difference in blastocyst development after intracy-
ner cells (Bruyas et al., 1993; Tremoleda et al., 2003; Hinrichs et al.,
toplasmic sperm injection (ICSI; Hinrichs et al., 2005).
2007b). The segregation of these inner cells is strikingly different
Standard IVF has not been reliably successful in the horse.
from that of other species: Enders et al. (1993) reported that some
Treatment of sperm to induce hyperactivation has resulted in
cells from this loose inner network form the ICM, and some mi-
>60% fertilization in two studies (McPartlin et al., 2009; Ambruosi
grate directly to individually seed the inside of the trophoblast,
et al., 2013). Currently, fertilization in the horse is performed using
then spread to form a continuous endodermal layer, resulting in
ICSI. Good blastocyst rates (20% to 40% of injected oocytes) are
a bilaminar blastocyst.
achieved using the Piezo drill (Fig. 7; Hinrichs et al., 2005; Galli
An acellular capsule forms inside the zona pellucida after entry
et al., 2007; Ribeiro et al., 2008). No exogenous activation is
of the equine embryo into the uterus (Flood et al., 1982; Freeman
needed.
et al., 1991). The equine capsule is composed of mucin-like glyco-
Low-glucose embryo culture media do not support equine blas-
proteins produced by the trophectoderm, containing a high pro-
tocyst formation. Good rates of blastocyst development have been
portion of sialic acid (Oriol et al., 1993a, 1993b). Sialic acid
achieved using DMEM/F-12 with fetal bovine serum in a mixed gas
transporters and sialyltransferases are upregulated from days 8
atmosphere (5% O2, 5% CO2, 90% N2), at 38.2 °C (Hinrichs et al.,
to 14 (Klein and Troedsson, 2011).
2005; Choi et al., 2007; Ribeiro et al., 2008). Equine embryos devel-
Guest and Allen (2007) found that FUT4 (previously known as
op to the blastocyst stage between days 7 and 10 after ICSI. Preg-
SSEA1), SSEA3, and SSEA4 proteins were expressed in both ICM
nancy rates after transfer of IVP blastocysts are 50–70% (Colleoni
and trophoblast in day-7 in vivo-recovered horse blastocysts,
et al., 2007; Choi et al., 2011). IVP is currently used clinically in
whereas POU5F1, TRA-1-60, TRA-1-81, and AP activity were local-
the horse, both in live mares (Colleoni et al., 2007) and post mor-
ized to the ICM. The ICM cells of Day 10 IVP/transferred embryos
tem (Hinrichs et al., 2012).
expressed significantly higher levels of SOX2 and NANOG than did
trophoblast; interestingly, CDX2 expression was present in both
Somatic cell nuclear transfer (SCNT)
cell types (Choi et al., 2009a) and has been reported in the equine
embryo proper at days 21–25 (de Mestre et al., 2009). Klein and
Woods et al. (2003) reported the birth of the first cloned equid,
Troedsson (2011) found that embryonic fibrinogen mRNA in-
a mule. Viable foals from SCNT have been reported from the labo-
creased from day 8 to day 14, and that fibrinogen was present in
ratory of Dr. Cesare Galli, in Italy (2 foals; Galli et al., 2003a, 2003b;
the conceptus and environs.
Lagutina et al., 2005), from our laboratory at Texas A&M (13 foals;
Smits et al. (2011) found five genes upregulated in equine
Choi et al., 2009b; Choi et al., 2013; Hinrichs et al., 2006, 2007a),
in vivo-derived vs. IVP blastocysts: FABP3, HSP90AA1, ODC1 (previ-
and from the laboratory of Dr. Daniel Salamone, in Argentina (2
ously known as ODC), MOB3 (previously known as MOBKL3), and
foals; Gambini et al., 2012). In addition, a company, ViaGen2 has
BEX2. Heat-shock protein HSPA1A mRNA was higher in IVP vs.
announced in the popular press the production of more than 160 via-
in vivo-derived embryos (Mortensen et al., 2010). Choi et al.
ble cloned foals.
(2009a) found that production of POU5F1 began at the com-
The reported blastocyst rate per reconstructed equine oocyte is
pacted-morula stage in IVP embryos. POU5F1 protein was limited
typically less than 10%. Reconstruction was performed by fusion
to the ICM in in vivo-derived embryos but not in IVP embryos,
with zona-free oocytes in Italy (Galli et al., 2003a, 2003b; Lagutina
and transfer of IVP embryos to the uterus normalized expression.
et al., 2005), and this technique, accompanied by aggregation of
Similarly, GATA6 protein was present only in hypoblast of day-
multiple reconstructed oocytes, was used in Argentina (Gambini
7.5 in vivo-derived embryos, but showed embryo-wide expression
et al., 2012). Our laboratory in Texas synchronizes donor cells with
in IVP embryos (Desmarais et al., 2011).
roscovitine, injects the cells into enucleated oocytes, and injects
At about day 37, specialized equine embryonic trophectoderm
sperm extract in addition to using chemical activation (Hinrichs
cells (chorionic girdle cells) invade into the maternal endome-
et al., 2006, 2007a; Choi et al., 2009b, 2013). Live foal production
trium, form nests (endometrial cups), and secrete equine chorionic
per embryo transferred reaches 35% (Hinrichs et al., 2007a). We re-
gonadotropin (eCG). Expression of GCM1, a transcription factor
ported on the health of cloned foals after birth (Johnson et al.,
found in human syncytiotrophoblast cells, was upregulated in cho-
2010). There was a 50% incidence of maladjustment, enlarged
rionic girdle cells at day 34 (de Mestre et al., 2009). Chorionic girdle
umbilical remnant, and/or front leg contracture. Two of 14 live-
cells also showed high expression of the immunoregulatory cyto-
born foals in this series died within 2 weeks of birth; the other
kine, interleukin (IL) 22, which may modulate endometrial re-
12 were viable. One of three foals in Italy died within 2 days of
sponse to invasion (Brosnahan et al., 2012).
birth (Lagutina et al., 2005). The two foals born in Argentina were
healthy (Gambini et al., 2012). No reports on foal viability are
In vitro production (IVP) of embryos available from ViaGen.

Immature equine oocytes may be recovered post mortem, (Hin-

richs and Williams, 1997; Hinrichs et al., 2005) or from live mares 2
See: www.ViaGen.com.
136 V. Hall et al. / The Veterinary Journal 197 (2013) 128–142

Embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC)

Saito et al. (2002) first described the production of equine ESC-

like cells after microsurgical dissection of ICM from day-8 horse
blastocysts. The cells expressed FUT4, STAT3, and POU5F1 and
could be differentiated to neural precursor cells. Li et al. (2006)
established ESC-like cells after immunosurgical dissection of ICM
from day-7 to -8 blastocysts; cells proliferated for up to 28 pas-
sages were positive for AP activity, and for FUT4, TRA-1-60, TRA-
1-81, and POU5F1 protein and mRNA. The cells differentiated into
multiple lineages but did not form teratomas when injected into
SCID mice. The gene pattern differed from those for mouse and hu-
man ES cells, but reflected the pattern of equine ICM cells (Guest
and Allen, 2007). Desmarais et al. (2011) produced ESC-like cells
from enzymatically-isolated ICM cells of in vivo-derived, partheno-
genetic, and SCNT embryos. These authors concluded the cells
most likely represented trophoblast stem cells rather than true
ESCs.
Nagy et al. (2011) first reported the generation of equine iPSCs,
after introducing human POU5F1, SOX2, KLF4, and MYC (also known
as c-MYC) into equine fetal fibroblasts. The resulting iPSCs ex-
pressed AP activity, FUT4, SSEA4, TRA-1-60, TRA-1-81, and NANOG,
as well as equine-specific mRNA for POU5F1, NANOG, and KLF4, and
formed teratomas. Breton et al. (2013) and Khodadadi et al. (2012)
reported production of iPSCs from adult equine fibroblasts; the lat-
ter study without use of MYC. Hackett et al. (2012) compared DNA
methylation patterns of the NANOG and SOX2 promoter regions
and concurrent gene expression of NANOG, SOX2, and POU5F1 in
equine iPSCs (Nagy Lab) with those of mesenchymal progenitor
cells (commercially marketed as ‘stem cells’ for treatment of ortho-
paedic injury in horses). All three pluripotency genes were highly
expressed in iPSCs, whereas mesenchymal progenitor cells ex-
pressed SOX2 at differentiated levels and did not express NANOG
or POU5F1. ESCs and iPSCs have extensive clinical application in
the treatment of orthopedic injury in horses, which presents an
excellent model for their use in the human athlete; thus, this is
an active and relatively well-funded area of research.

Dog (Canis lupus familiaris)

Molecular regulation of pre-implantation development

Several characteristics of canine female reproductive physiol-

ogy and developmental biology, such as a pre-ovulatory follicular
luteinization, ovulation of immature germinal vesicle (GV) stage
oocytes, and prolonged pre-implantation development/transport
in the oviduct are distinctly different from those seen in other
mammalian species (Reynaud et al., 2006). These differences may
account for the very poor efficiency of IVM, IVF, and IVC in canines
(Chastant-Maillard et al., 2010). One unique feature of canine oo-
cytes, zygotes, and embryos is their abundant lipid content that
accumulates during follicle growth (Tesoriero, 1981). These lipids,
Fig. 7. Equine in vitro production of embryos. Equine in vitro matured oocyte (A), 8- mainly made up of triglycerides and phospholipids, are clearly vis-
cell embryo derived from intracytoplasmic sperm injection (B), and expanded
ible by microscopy and can still be observed in the ICM and troph-
blastocyst (C). Note the lack of a distinct inner cell mass in (C). 1: Cumulus cells; 2:
Zona pellucida; 3: First polar body. Scale bars: 50 lm (A, B, and C). ectoderm of canine blastocysts (Fig. 8) and may be indicative of a
unique metabolism that could contribute to their poor develop-
ment in current in vitro regimes (see below).
Because of these hurdles very limited studies have examined
the molecular regulation of canine pre-implantation development.
In day-10 flushed canine embryos (morulae and early blastocysts)
Equine cloning presents an excellent tool for research on genet- transcripts for key enzymes of prostaglandin synthesis (COX2), se-
ics vs. environment in a host of equine diseases, but has not yet lected growth factors (TGF-a, IGF-1, 2), cytokines (IL-1a, 6), im-
been utilized for such studies. Commercial equine cloning is per- mune cell receptors (CD4) and matrix-metalloproteinases (MMP-2
formed to preserve valuable genetics, but clones and their offspring and 9) are present (Schafer-Somi et al., 2008). We have detected
are not eligible for registration in most breeds. early lineage markers of the epiblast (POU5F1), trophectoderm
 V. Hall et al. / The Veterinary Journal 197 (2013) 128–142 137

Somatic cell nuclear transfer (SCNT)

While poor rates of IVM and IVF have limited canine ART (Cha-
stant-Maillard et al., 2010), SCNT could be used to improve canine
reproduction and produce valuable disease models. Since the birth
of ‘Snuppy’, the first cloned dog (Lee et al., 2005), several breeds of
viable cloned puppies have now been produced by SCNT (Jang
et al., 2008; Hossein et al., 2009; Kim et al., 2012). Due to the dog’s
unique reproductive physiology, limitations of canine in vitro em-
bryo technologies (see above) and poor ovulation induction, dog
cloning has relied heavily on protocols to surgically collect
in vivo matured oocytes by oviduct flushing after predicted natural
ovulations (Johnston et al., 2001; Lee et al., 2005) and surgically
transfer cloned embryos immediately after reconstruction to spon-
taneously synchronized recipients (Lee et al., 2005). Nevertheless,
over 50 cloned dogs have been reported with an average pregnancy
rate of almost 18% and an average live birth rate of 1.42% from total
number of embryos transferred (Kim et al., 2012).
Cloning can propagate desired canine traits, restore the repro-
ductive ability of old or neutered dogs, and even ‘resurrect’ a dead
pet (Jang et al., 2008; Park et al., 2009). The concept of dog cloning
was fostered in 1998 with the multi-million dollar Missyplicity
project designed to clone a dog called ‘Missy’. Although initially
Fig. 8. Expanded canine blastocyst derived in vivo. At 11–15 days following unsuccessful, Missy’s clone was born in 2007 as the World’s first
detection of an LH surge canine blastocysts were retrieved from flushed reproduc- clone of a family dog. Currently, there are a few companies (e.g.
tive tracts of inseminated bitches. Note the dark lipid-rich inner cell mass (1). 2:
RNL Bio; Perpetuate) that commercially offer canine cloning ser-
Zona pellucida; 3: Blastocyst cavity. Scale bar: 100 lm.
vices and/or will cryobank cells for producing future dog clones
when the technology becomes more efficient and cheaper.
(CDX2), and hypoblast (GATA6) in flushed canine blastocysts Although phenotypic differences have been observed between
(Wilcox et al., 2009). Like other domestic species (Kirchhof et al., clones due to stochastic epigenetic reprogramming events (Peat
2000), POU5F1 is expressed at diminished levels in the and Reik, 2012), SCNT allows elite characteristics related to nuclear
trophectoderm (Wilcox et al., 2009). Clearly, further fundamental genetic information to be passed on to the clones. Since re-cloned
research is still required to understand the molecular mechanisms dogs have been recently derived from cells of cloned canines (Hong
governing oocyte maturation and embryonic development in the et al., 2011b; Oh et al., 2011), infinite propagation of these elite
dog. abilities, such as the unique scent sniffing capabilities of detection
dogs (Park et al., 2009) or even transgenic dogs (Hong et al., 2009;
Kim et al., 2011) are theoretically possible. Interspecies SCNT has
In vitro production (IVP) of embryos even been used for preservation of endangered canine species.
The grey wolf (Canis lupus), which is considered a threatened spe-
Although conventional assisted reproductive technologies such cies in many countries, was successfully cloned using a wolf so-
as AI and cryopreservation have been highly successful (Thomas- matic cell and a dog oocyte and recipient (Kim et al., 2007; Oh
sen and Farstad, 2009), other advanced technologies have been et al., 2008).
exceptionally inadequate for obtaining high rates of in vitro em- Owing to a shared environment and to similarities in physiol-
bryo development in the dog (Chastant-Maillard et al., 2010). For ogy, disease presentation, and clinical response at least half of ca-
starters, the IVM rates of canine oocytes are very low compared nine diseases are known to have human equivalents making the
to results obtained in other domestic species (Otoi et al., 2000; Gal- dog an ideal model for human disorders (Starkey et al., 2005).
li and Lazzari, 2008; Bukowska et al., 2012). Canine oocytes col- Although controversial (Varner, 1999; Fiester, 2005), using SCNT
lected from anoestrous ovaries exhibit very low frequencies (10– to generate genetically modified disease models in dogs looks
20%) of maturation to the MII stage after 72–96 h of culture (Luv- promising (Jeong et al., 2012; Oh et al., 2012). However, as with
oni et al., 2005; Songsasen and Wildt, 2007), while the IVM rate of other domestic animal clones (Wells, 2005), there have also been
oocytes from pre-ovulatory follicles only reaches about 30% (Yam- some reports of abnormalities in cloned dogs that would currently
ada et al., 1993). limit this use (Kim et al., 2009; Hong et al., 2011a). These reports
Compounding this poor IVM is a reduced ability of canine sper- are contentious, however, since there are other published studies
matozoa to penetrate (10–50%) these oocytes in vitro, with only 4– showing no adverse effects (Hong et al., 2010; Park et al., 2010).
10% of all oocytes forming two pronuclei after IVF (Mahi and Yan- Nevertheless, the challenging reproductive physiological barriers
agimachi, 1976; De los Reyes et al., 2009). The poor fertilization is combined with poor ART, low SCNT efficiency, and the high costs
due, in part, to high rates of polyspermy (Saint-Dizier et al., 2001; associated with these technologies are still limiting factors for
Hatoya et al., 2006a). ICSI has equally been poor and has not over- achieving translational success with dog cloning.
come this dual problem of polyspermy and low fertilization ability
(Fulton et al., 1998). Although we observed decent in vitro devel- Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs)
opment of in vivo fertilized and flushed canine morulae to the blas-
tocyst stages in SOF medium cultured under 5% oxygen tensions One emerging research field that may contribute to a greater
(Wilcox et al., 2009), a 4- to 8-cell block commonly occurs that understanding of pre-implantation embryonic development in
contributes to their exceptionally poor in vitro development (Yam- general and reveal the dog as a model system for developing ther-
ada et al., 1992; Otoi et al., 2000; Hori and Tsutsui, 2003; Hatoya apeutic treatments is canine pluripotent stem cells. We were
et al., 2006a). among five research groups to separately derive the first canine
138 V. Hall et al. / The Veterinary Journal 197 (2013) 128–142

Canine pluripotent stem cells may exist in a stably distinct plu-

ripotent state. However, preliminary studies in our laboratory have
shown that canine ESCs are responsive to LIF and 2i (inhibitors of
the Mek/Erk and GSK3 pathways) supplemented media with colo-
nies displaying domed-like morphology typical of naïve pluripo-
tency (Fig. 9). Further examination of these unique canine ESCs
and iPSCs will allow us to understand to a greater extent the origin
of different pluripotent states and will help define/optimize spe-
cific culture conditions for their unlimited self-renewal and differ-
entiation into therapeutically relevant cell types to create animal
models, stem cell transplantation treatments, and/or as drug
screening modalities for human/canine diseases.

Conclusions

Over the coming years, because of new technological advance-

ments and transcriptome- and proteome-based insight into cellu-
lar molecular signalling pathways, further progress in embryo
and stem cell culture conditions will be forthcoming. This im-
proved efficiency will increase the safety, efficacy and applicability
of assisted reproductive technologies in animal production. How-
ever, the greatest impact is expected to be in the area of biomedi-
cine. As with the human stem-cell field, advancements in domestic
animal stem cells, and, in particular in iPSCs, will create cellular
and animal models of disease that may also be used as drug-
screening tools to treat various livestock and companion-animal
ailments. In combination with newly-discovered genome editing
tools, possible cell-based therapies to regenerate, repair, or even
replace damaged or diseased tissue are envisioned in the near
future.

Conflict of interest statement

Fig. 9. Naïve (A) and primed (B) canine embryonic stem cell colonies. Canine ESC None of the authors has any financial or personal relationships
colonies appear in a primed (hESC-like) pluripotent state when cultured in media that could inappropriately influence or bias the content of the
contain LIF and bFGF, but morphological changes associated with a more naïve
paper.
(mESC-like) pluripotent state are observed upon propagation of explanted colonies
in media containing just LIF + 2i (glycogen synthase kinase 3b and mitogen-
activated protein kinase inhibitors). Scale bars: 100 lm (A and B).
Acknowledgements

We would like to thank for the financial support from the EU

ESC lines (Hatoya et al., 2006b; Schneider et al., 2007; Hayes et al., projects EU FP7 PartnErS, PIAP-GA-2008-218205 and PluriSys,
2008; Vaags et al., 2009; Wilcox et al., 2009). All of our character- HEALTH-2007-B-223485 as well as from the Danish National Ad-
ized lines exhibit prolonged propagation in the undifferentiated vanced Technology Foundation and The Danish Council for Inde-
state by the expression of established pluripotency markers (e.g. pendent Research / Natural Sciences.
POU5F1, SOX2, and SSEA3). Under culture conditions supporting
differentiation, canine ESCs form embryoid bodies that give rise
References
to ectodermal, mesodermal, and endodermal derivatives (Wilcox
et al., 2009). Importantly, we have recently established neural pro- Abeydeera, L.R., 2002. In vitro production of embryos in swine. Theriogenology 57,
genitors and active neural cell types from our canine ESC lines 256–273.
Algriany, O., Bevers, M., Schoevers, E., Colenbrander, B., Dieleman, S., 2004. Follicle
(Wilcox et al., 2011). Although chimera formation and germ line
size-dependent effects of sow follicular fluid on in vitro cumulus expansion,
contribution of canine ESCs have not yet been assessed, some nuclear maturation and blastocyst formation of sow cumulus oocytes
ESC lines have displayed small overt teratoma formation after complexes. Theriogenology 62, 1483–1497.
Ambruosi, B., Accogli, G., Douet, C., Canepa, S., Pascal, G., Monget, P., Moros Nicolas,
transplantation into immune compromised mice (Vaags et al.,
C., Holmskov, U., Mollenhauer, J., Robbe-Masselot, C., Vidal, O., Desantis, S.,
2009; Wilcox et al., 2009). Goudet, G., 2013. Deleted in malignant brain tumour 1 (DMBT1) is secreted in
Different pluripotent states (naïve vs. primed) likely arise from the oviduct and involved in the mechanism of fertilization in equine and
the species-specific developmental stage from which the cells are porcine species. Reproduction. http://dx.doi.org/10.1530/REP-13-0007.
Anderson, J.E., Matteri, R.L., Abeydeera, L.R., Day, B.N., Prather, R.S., 2001.
derived (Nichols and Smith, 2009) and stabilized by the in vitro con- Degradation of maternal cdc25c during the maternal to zygotic transition is
ditions they are cultured in (Hanna et al., 2009). Although canine dependent upon embryonic transcription. Molecular Reproduction and
ESCs and canine iPSCs have been derived in LIF-only medium (Hayes Development 60, 181–188.
Brackett, B.G., Bousquet, D., Boice, M.L., Donawick, W.J., Evans, J.F., Dressel, M.A.,
et al., 2008; Whitworth et al., 2012), their long term propagation un- 1982. Normal development following in vitro fertilization in the cow. Biology of
der these conditions have not been demonstrated. Interestingly, it Reproduction 27, 147–158.
appears that canine ESCs (Vaags et al., 2009; Wilcox et al., 2009) Breton, A., Sharma, R., Diaz, A.C., Parham, A.G., Graham, A., Neil, C., Whitelaw, C.B.,
Milne, E., Donadeu, F.X., 2013. Derivation and characterization of induced
and iPSCs (Luo et al., 2011) require dual-factor culture (LIF and bFGF) pluripotent stem cells from equine fibroblasts. Stem Cells and Development 22,
to maintain proliferation in the undifferentiated state. 611–621.
 V. Hall et al. / The Veterinary Journal 197 (2013) 128–142 139

Brinsko, S.P., Ball, B.A., Ignotz, G.G., Thomas, P.G.A., Currie, W.B., Ellington, J.E., 1995. Degrelle, S.A., Campion, E., Cabau, C., Piumi, F., Reinaud, P., Richard, C., Renard, J.P.,
Initiation of transcription and nucleologenesis in equine embryos. Molecular Hue, I., 2005. Molecular evidence for a critical period in mural trophoblast
Reproduction and Development 42, 298–302. development in bovine blastocysts. Developmental Biology 288, 448–460.
Brons, I.G., Smithers, L.E., Trotter, M.W., Rugg-Gunn, P., Sun, B., Chuva de Sousa Desmarais, J.A., Demers, S.P., Suzuki Jr., J., Laflamme, S., Vincent, P., Laverty, S.,
Lopes, S.M., Howlett, S.K., Clarkson, A., Ahrlund-Richter, L., Pedersen, R.A., Smith, L.C., 2011. Trophoblast stem cell marker gene expression in inner cell
Vallier, L., 2007. Derivation of pluripotent epiblast stem cells from mammalian mass-derived cells from parthenogenetic equine embryos. Reproduction 141,
embryos. Nature 448, 191–195. 321–332.
Brosnahan, M.M., Miller, D.C., Adams, M., Antczak, D.F., 2012. IL-22 is expressed by Enders, A.C., Schlafke, S., Lantz, K.C., Liu, I.K.M., 1993. Endoderm cells of the equine
the invasive trophoblast of the equine (Equus caballus) chorionic girdle. Journal yolk sac from day 7 until formation of the definitive yolk sac placenta. Equine
of Immunology 188, 4181–4187. Veterinary Journal Supplement 15, 3–9.
Brück, I., Raun, K., Synnestvedt, B., Greve, T., 1992. Follicle aspiration in the mare Evans, M.J., Kaufman, M.H., 1981. Establishment in culture of pluripotential cells
using a transvaginal ultrasound-guided technique (short communication). from mouse embryos. Nature 292, 154–156.
Equine Veterinary Journal 24, 58–59. Everts, R.E., Chavatte-Palmer, P., Razzak, A., Hue, I., Green, C.A., Oliveira, R., Vignon,
Bruyas, J.F., Bezard, J., Lagneaux, D., Palmer, E., 1993. Quantitative analysis of X., Rodriguez-Zas, S.L., Tian, X.C., Yang, X., Renard, J.P., Lewin, H.A., 2008.
morphological modifications of day 6.5 horse embryos after cryopreservation: Aberrant gene expression patterns in placentomes are associated with
Differential effects on inner cell mass and trophoblast cells. Journal of phenotypically normal and abnormal cattle cloned by somatic cell nuclear
Reproduction and Fertility 99, 15–23. transfer. Physiology Genomics 33, 65–77.
Buehr, M., Meek, S., Blair, K., Yang, J., Ure, J., Silva, J., McLay, R., Hall, J., Ying, Q.L., Ezashi, T., Telugu, B.P., Roberts, R.M., 2012. Induced pluripotent stem cells from pigs
Smith, A., 2008. Capture of authentic embryonic stem cells from rat blastocysts. and other ungulate species: An alternative to embryonic stem cells?
Cell 135, 1287–1298. Reproduction in Domestic Animals (Zuchthygiene) 47, 92–97.
Buhi, W.C., Alvarez, I.M., Kouba, A.J., 1997. Oviductal regulation of fertilization and Fiester, A., 2005. Ethical issues in animal cloning. Perspectives in Biological
early embryonic development. Journal of Reproduction and Fertility Suppl. 52, Medicine 48, 328–343.
285–300. Flood, P.F., Betteridge, K.J., Diocee, M.S., 1982. Transmission electron microscopy of
Bukowska, D., Kempisty, B., Piotrowska, H., Zawierucha, P., Brussow, K.P., Jaskowski, horse embryos 3–16 days after ovulation. Journal of Reproduction and Fertilility
J.M., Nowicki, M., 2012. The in vitro culture supplements and selected aspects of Suppl. 32, 319–327.
canine oocytes maturation. Polish Journal of Veterinary Sciences 15, 199–205. Freeman, D.A., Weber, J.A., Geary, R.T., Woods, G.L., 1991. Time of embryo transport
Campbell, K.H., Loi, P., Otaegui, P.J., Wilmut, I., 1996. Cell cycle co-ordination in through the mare oviduct. Theriogenology 36, 823–830.
embryo cloning by nuclear transfer. Reviews of Reproduction 1, 40–46. Fujimura, T., Murakami, H., Kurome, M., Takahagi, Y., Shigehisa, T., Nagashima, H.,
Cao, H., Yang, P., Pu, Y., Sun, X., Yin, H., Zhang, Y., Li, Y., Liu, Y., Fang, F., Zhang, Z., Tao, 2008. Effects of recloning on the efficiency of production of alpha 1,3-
Y., Zhang, X., 2012. Characterization of bovine induced pluripotent stem cells by galactosyltransferase knockout pigs. The Journal of Reproduction and
lentiviral transduction of reprogramming factor fusion proteins. International Development 54, 58–62.
Journal of Biological Science 8, 498–511. Fulton, R.M., Keskintepe, L., Durrant, B.S., Fayrer-Hosken, R.A., 1998.
Carnevale, E.M., Ginther, O.J., 1993. Use of a linear ultrasonic transducer for the Intracytoplasmic sperm injection (ICSI) for the treatment of canine infertility.
transvaginal aspiration and transfer of oocytes in the mare. Journal of Equine Theriogenology 49, 366.
Veterinary Science 13, 331–333. Galli, C., Lazzari, G., 2008. The manipulation of gametes and embryos in farm
Chastant-Maillard, S., Chebrout, M., Thoumire, S., Saint-Dizier, M., Chodkiewicz, M., animals. Reproduction in Domestic Animals 43, 1–7.
Reynaud, K., 2010. Embryo biotechnology in the dog: A review. Reproduction Galli, C., Duchi, R., Moor, R.M., Lazzari, G., 1999. Mammalian leukocytes contain all
Fertility and Development 22, 1049–1056. the genetic information necessary for the development of a new individual.
Choi, Y.H., Love, L.B., Varner, D.D., Hinrichs, K., 2006. Holding immature equine Cloning 1, 161–170.
oocytes in the absence of meiotic inhibitors: Effect on germinal vesicle Galli, C., Duchi, R., Crotti, G., Turini, P., Ponderato, N., Colleoni, S., Lagutina, I., Lazzari,
chromatin and blastocyst development after intracytoplasmic sperm G., 2003a. Bovine embryo technologies. Theriogenology 59, 599–616.
injection. Theriogenology 66, 955–963. Galli, C., Lagutina, I., Crotti, G., Colleoni, S., Turini, P., Ponderato, N., Duchi, R., Lazzari,
Choi, Y.H., Love, L.B., Varner, D.D., Hinrichs, K., 2007. Effect of holding technique and G., 2003b. A cloned horse born to its dam twin. Nature 424, 635.
culture drop size in individual or group culture on blastocyst development after Galli, C., Colleoni, S., Duchi, R., Lagutina, I., Lazzari, G., 2007. Developmental
ICSI of equine oocytes with low meiotic competence. Animal Reproduction competence of equine oocytes and embryos obtained by in vitro procedures
Science 102, 38–47. ranging from in vitro maturation and ICSI to embryo culture, cryopreservation
Choi, Y.H., Harding, H.D., Hartman, D.L., Obermiller, A.D., Kurosaka, S., McLaughlin, and somatic cell nuclear transfer. Animal Reproduction Science 98, 39–55.
K.J., Hinrichs, K., 2009a. The uterine environment modulates trophectodermal Gambini, A., Jarazo, J., Olivera, R., Salamone, D.F., 2012. Equine cloning: In vitro and
POU5F1 levels in equine blastocysts. Reproduction 138, 589–599. in vivo development of aggregated embryos. Biology of Reproduction 87, 11–19.
Choi, Y.H., Hartman, D.L., Fissore, R.A., Bedford-Guaus, S.J., Hinrichs, K., 2009b. Effect Gao, Y., Hyttel, P., Hall, V.J., 2010. Regulation of H3K27me3 and H3K4me3 during
of sperm extract injection volume, injection of PLCzeta cRNA, and tissue cell line early porcine embryonic development. Molecular Reproduction and
on efficiency of equine nuclear transfer. Cloning and Stem Cells 11, 301–308. Development 77, 540–549.
Choi, Y.H., Varner, D.D., Love, C.C., Hartman, D.L., Hinrichs, K., 2011. Production of Gao, Y., Hyttel, P., Hall, V.J., 2011a. Dynamic changes in epigenetic marks and gene
live foals via intracytoplasmic injection of lyophilized sperm and sperm extract expression during porcine epiblast specification. Cellular Reprogramming 13,
in the horse. Reproduction 142, 529–538. 345–360.
Choi, Y.H., Norris, J.D., Velez, I.C., Jacobson, C.C., Hartman, D.L., Hinrichs, K., 2013. A Gao, Y., Jammes, H., Rasmussen, M.A., Oestrup, O., Beaujean, N., Hall, V., Hyttel, P.,
viable foal obtained by equine somatic cell nuclear transfer using oocytes 2011b. Epigenetic regulation of gene expression in porcine epiblast, hypoblast,
recovered from immature follicles of live mares. Theriogenology 79, 791–796. trophectoderm and epiblast-derived neural progenitor cells. Epigenetics 6,
Cibelli, J.B., Stice, S.L., Golueke, P.J., Kane, J.J., Jerry, J., Blackwell, C., Ponce de Leon, 1149–1161.
F.A., Robl, J.M., 1998a. Cloned transgenic calves produced from nonquiescent Gardner, D.K., Lane, M., Spitzer, A., Batt, P.A., 1994. Enhanced rates of cleavage and
fetal fibroblasts. Science 280, 1256–1258. development for sheep zygotes cultured to the blastocyst stage in vitro in the
Cibelli, J.B., Stice, S.L., Golueke, P.J., Kane, J.J., Jerry, J., Blackwell, C., Ponce de Leon, absence of serum and somatic cells: Amino acids, vitamins, and culturing
F.A., Robl, J.M., 1998b. Transgenic bovine chimeric offspring produced from embryos in groups stimulate development. Biology of Reproduction 50, 390–
somatic cell-derived stem-like cells. Nature Biotechnology 16, 642–646. 400.
Colleoni, S., Barbacini, S., Necci, D., Duchi, R., Lazzari, G., Galli, C., 2007. Application Gil, M.A., Cuello, C., Parrilla, I., Vazquez, J.M., Roca, J., Martinez, E.A., 2010. Advances
of ovum pick-up, intracytoplasmic sperm injection and embryo culture in in swine in vitro embryo production technologies. Reproduction in Domestic
equine practice. In: Proceedings of the American Association of Equine Animals (Zuchthygiene) 45, 40–48.
Practitioners, pp. 554–559. Gjorret, J.O., Maddox-Hyttel, P., 2005. Attempts towards derivation and
Coy, P., Grullon, L., Canovas, S., Romar, R., Matas, C., Aviles, M., 2008. Hardening of establishment of bovine embryonic stem cell-like cultures. Reproduction,
the zona pellucida of unfertilized eggs can reduce polyspermic fertilization in Fertility and Development 17, 113–124.
the pig and cow. Reproduction 135, 19–27. Grondahl, C., Hyttel, P., 1996. Nucleologenesis and ribonucleic acid synthesis in
Day, B.N., 2000. Reproductive biotechnologies: Current status in porcine preimplantation equine embryos. Biology of Reproduction 55, 769–774.
reproduction. Animal Reproduction Science 60–61, 161–172. Guest, D.J., Allen, W.R., 2007. Expression of cell-surface antigens and embryonic
De Los Angeles, A., Loh, Y.H., Tesar, P.J., Daley, G.Q., 2012. Accessing naive human stem cell pluripotency genes in equine blastocysts. Stem Cells and Development
pluripotency. Current Opinion in Genetics and Development 22, 272–282. 16, 789–796.
De los Reyes, M., Palomino, J., de Lange, J., Anguita, C., Barros, C., 2009. In vitro Hackett, C.H., Greve, L., Novakofski, K.D., Fortier, L.A., 2012. Comparison of gene-
sperm penetration through the zona pellucida of immature and in vitro specific DNA methylation patterns in equine induced pluripotent stem cell lines
matured oocytes using fresh, chilled and frozen canine semen. Animal with cells derived from equine adult and fetal tissues. Stem Cells and
Reproduction Science 110, 37–45. Development 21, 1803–1811.
de Mestre, A.M., Miller, D., Roberson, M.S., Liford, J., Chizmar, L.C., McLaughlin, K.E., Hall, V., 2008. Porcine embryonic stem cells: A possible source for cell replacement
Antczak, D.F., 2009. Glial cells missing homologue 1 is induced in differentiating therapy. Stem Cell Reviews 4, 275–282.
equine chorionic girdle trophoblast cells. Biology of Reproduction 80, 227–234. Hall, V.J., 2013. Early development of the porcine embryo: The importance of cell
Dean, W., Santos, F., Stojkovic, M., Zakhartchenko, V., Walter, J., Wolf, E., Reik, W., signaling in development of pluripotent cell lines. Reproduction Fertility and
2001. Conservation of methylation reprogramming in mammalian Development 25, 94–102.
development: Aberrant reprogramming in cloned embryos. Proceedings of the Hall, V.J., Christensen, J., Gao, Y., Schmidt, M.H., Hyttel, P., 2009. Porcine
National Academy of Sciences USA 98, 13734–13738. pluripotency cell signaling develops from the inner cell mass to the epiblast
140 V. Hall et al. / The Veterinary Journal 197 (2013) 128–142

during early development. Developmental Dynamics: An Official Publication of Hyttel, P., Sinowatz, F., Vejlsted, M., 2009. Essentials of Domestic Animal
the American Association of Anatomists 238, 2014–2024. Embryology. Elsevier Science, 455 pp. ISBN:978-0-7020-2899-1.
Hall, V.J., Kristensen, M., Rasmussen, M.A., Ujhelly, O., Dinnyes, A., Hyttel, P., 2012. Iwasaki, S., Campbell, K.H., Galli, C., Akiyama, K., 2000. Production of live calves
Temporal repression of endogenous pluripotency genes during reprogramming derived from embryonic stem-like cells aggregated with tetraploid embryos.
of porcine induced pluripotent stem cells. Cellular Reprogramming 14, 204– Biology of Reproduction 62, 470–475.
216. Jacobson, C.C., Choi, Y.H., Hayden, S.S., Hinrichs, K., 2010. Recovery of mare oocytes
Hanna, J., Markoulaki, S., Mitalipova, M., Cheng, A.W., Cassady, J.P., Staerk, J., Carey, on a fixed biweekly schedule, and resulting blastocyst formation after
B.W., Lengner, C.J., Foreman, R., Love, J., Gao, Q., Kim, J., Jaenisch, R., 2009. intracytoplasmic sperm injection. Theriogenology 73, 1116–1126.
Metastable pluripotent states in NOD-mouse-derived ESCs. Cell Stem Cell 4, Jang, G., Hong, S.G., Oh, H.J., Kim, M.K., Park, J.E., Kim, H.J., Kim, D.Y., Lee, B.C., 2008.
513–524. A cloned toy poodle produced from somatic cells derived from an aged female
Hatoya, S., Sugiyama, Y., Torii, R., Wijewardana, V., Kumagai, D., Sugiura, K., Kida, K., dog. Theriogenology 69, 556–563.
Kawate, N., Tamada, H., Sawada, T., Inaba, T., 2006a. Effect of co-culturing with Jarrell, V.L., Day, B.N., Prather, R.S., 1991. The transition from maternal to zygotic
embryonic fibroblasts on IVM, IVF and IVC of canine oocytes. Theriogenology control of development occurs during the 4-cell stage in the domestic pig, Sus
66, 1083–1090. scrofa: Quantitative and qualitative aspects of protein synthesis. Biology of
Hatoya, S., Torii, R., Kondo, Y., Okuno, T., Kobayashi, K., Wijewardana, V., Kawate, N., Reproduction 44, 62–68.
Tamada, H., Sawada, T., Kumagai, D., Sugiura, K., Inaba, T., 2006b. Isolation and Jeong, Y.W., Lee, G.S., Kim, J.J., Park, S.W., Ko, K.H., Kang, M., Kim, Y.K., Jung, E.M.,
characterization of embryonic stem-like cells from canine blastocysts. Hyun, S.H., Shin, and others, 2012. Establishment of a canine model of human
Molecular Reproduction and Development 73, 298–305. type 2 diabetes mellitus by overexpressing phosphoenolypyruvate
Hayes, B., Fagerlie, S.R., Ramakrishnan, A., Baran, S., Harkey, M., Graf, L., Bar, M., carboxykinase. International Journal on Molecular Medicine 30, 321–329.
Bendoraite, A., Tewari, M., Torok-Storb, B., 2008. Derivation, characterization, Johnson, A.K., Clark-Price, S.C., Choi, Y.H., Hartman, D.L., Hinrichs, K., 2010. Physical
and in vitro differentiation of canine embryonic stem cells. Stem Cells (Dayton, and clinicopathologic findings in foals derived by use of somatic cell nuclear
Ohio) 26, 465–473. transfer: 14 cases (2004–2008). Journal of the American Veterinary Medical
Heyman, Y., Chavatte-Palmer, P., LeBourhis, D., Camous, S., Vignon, X., Renard, J.P., Association 236, 983–990.
2002a. Frequency and occurrence of late-gestation losses from cattle cloned Johnston, S.D., Root Kustritz, M.V., Olson, P.N.S., 2001. The canine estrous cycle. In:
embryos. Biology of Reproduction 66, 6–13. Kersey, R., LeMelledo, D. (Eds.), Canine and Feline Theriogenology. WB Saunders
Heyman, Y., Richard, C., Rodriguez-Martinez, H., Lazzari, G., Chavatte-Palmer, P., Company, Philadeliphia, PA, pp. 16–31.
Vignon, X., Galli, C., 2004. Zootechnical performance of cloned cattle and Keefer, C.L., Pant, D., Blomberg, L., Talbot, N.C., 2007. Challenges and prospects for
offspring: Preliminary results. Cloning and Stem Cells 6, 111–120. the establishment of embryonic stem cell lines of domesticated ungulates.
Heyman, Y., Chavatte-Palmer, P., Fromentin, G., Berthelot, V., Jurie, C., Bas, P., Animal Reproduction Science 98, 147–168.
Dubarry, M., Mialot, J.P., Remy, D., Richard, C., Martignat, L., Vignon, X., Renard, Khan, D.R., Dube, D., Gall, L., Peynot, N., Ruffini, S., Laffont, L., Le Bourhis, D., Degrelle,
J.P., 2007. Quality and safety of bovine clones and their products. Animal 1, S., Jouneau, A., Duranthon, V., 2012. Expression of pluripotency master
963–972. regulators during two key developmental transitions: EGA and early lineage
Hinrichs, K., Williams, K.A., 1997. Relationships among oocyte-cumulus specification in the bovine embryo. PLoS One 7, e34110.
morphology, follicular atresia, initial chromatin configuration, and oocyte Khodadadi, K., Sumer, H., Pashaiasl, M., Lim, S., Williamson, M., Verma, P.J., 2012.
meiotic competence in the horse. Biology of Reproduction 57, 377–384. Induction of pluripotency in adult equine fibroblasts without c-MYC. Stem Cells
Hinrichs, K., Matthews, G.L., Freeman, D.A., Torello, E.M., 1998. Oocyte transfer in International. http://dx.doi.org/10.1155/2012/429160, Epub..
mares. Journal of the American Veterinary Medical Association 212, 982–986. Kikuchi, K., Onishi, A., Kashiwazaki, N., Iwamoto, M., Noguchi, J., Kaneko, H., Akita,
Hinrichs, K., Choi, Y.H., Love, L.B., Varner, D.D., Love, C.C., Walckenaer, B.E., 2005. T., Nagai, T., 2002. Successful piglet production after transfer of blastocysts
Chromatin configuration within the germinal vesicle of horse oocytes: Changes produced by a modified in vitro system. Biology of Reproduction 66, 1033–
post mortem and relationship to meiotic and developmental competence. 1041.
Biology of Reproduction 72, 1142–1150. Kim, S.H., Li, C., Maller, J.L., 1999. A maternal form of the phosphatase Cdc25A
Hinrichs, K., Choi, Y.H., Love, C.C., Chung, Y.G., Varner, D.D., 2006. Production of regulates early embryonic cell cycles in Xenopus laevis. Developmental Biology
horse foals via direct injection of roscovitine-treated donor cells and activation 212, 381–391.
by injection of sperm extract. Reproduction 131, 1063–1072. Kim, M.K., Jang, G., Oh, H.J., Yuda, F., Kim, H.J., Hwang, W.S., Hossein, M.S., Kim, J.J.,
Hinrichs, K., Choi, Y.H., Varner, D.D., Hartman, D.L., 2007a. Production of cloned Shin, N.S., Kang, S.K., Lee, B.C., 2007. Endangered wolves cloned from adult
horse foals using roscovitine-treated donor cells and activation with sperm somatic cells. Cloning and Stem Cells 9, 130–137.
extract and/or ionomycin. Reproduction 134, 319–325. Kim, S., Park, S.W., Hossein, M.S., Jeong, Y.W., Kim, J.J., Lee, E., Kim, Y.W., Hyun, S.H.,
Hinrichs, K., Choi, Y.H., Walckenaer, B.E., Varner, D.D., Hartman, D.L., 2007b. In vitro- Shin, T., Hwang, W.S., 2009. Production of cloned dogs by decreasing the
produced equine embryos: Production of foals after transfer, assessment by interval between fusion and activation during somatic cell nuclear transfer.
differential staining, and effect of medium calcium concentrations during Molecular Reproduction and Development 76, 483–489.
culture. Theriogenology 68, 521–529. Kim, M.J., Oh, H.J., Park, J.E., Kim, G.A., Hong, S.G., Jang, G., Kwon, M.S., Koo, B.C., Kim,
Hinrichs, K., Choi, Y.H., Norris, J.D., Love, L.B., Bedford-Guaus, S.J., Hartman, D.L., T., Kang, S.K., and others, 2011. Generation of transgenic dogs that conditionally
Velez, I.C., 2012. Evaluation of foal production following intracytoplasmic express green fluorescent protein. Genesis 49, 472–478.
sperm injection and blastocyst culture of oocytes from ovaries collected Kim, M.J., Oh, H.J., Kim, G.A., Park, J.E., Park, E.J., Jang, G., Ra, J.C., Kang, S.K., Lee, B.C.,
immediately before euthanasia or after death of mares under field conditions. 2012. Lessons learned from cloning dogs. Reproduction in Domestic Animals
Journal of the American Veterinary Medical Association 241, 1070–1074. (Zuchthygiene) 47, 115–119.
Hong, S.G., Kim, M.K., Jang, G., Oh, H.J., Park, J.E., Kang, J.T., Koo, O.J., Kim, T., Kwon, Kirchhof, N., Carnwath, J.W., Lemme, E., Anastassiadis, K., Scholer, H., Niemann, H.,
M.S., Koo, B.C., and others, 2009. Generation of red fluorescent protein 2000. Expression pattern of Oct-4 in preimplantation embryos of different
transgenic dogs. Genesis 47, 314–322. species. Biology of Reproduction 63, 1698–1705.
Hong, S.G., Oh, H.J., Park, J.E., Kang, J.T., Kim, M.J., Yoon, J.H., Chang, J.H., Kim, M.K., Klein, C., Troedsson, M.H., 2011. Transcriptional profiling of equine conceptuses
Jang, G., Lee, B.C., 2010. Serum levels of reproductive hormones and reveals new aspects of embryo-maternal communication in the horse. Biology
ultrasonographic monitoring of ovarian follicles in female cloned dogs. of Reproduction 84, 872–885.
Journal of Veterinary Medical Science 72, 89–92. Klymiuk, N., Mundhenk, L., Kraehe, K., Wuensch, A., Plog, S., Emrich, D.,
Hong, I.H., Jeong, Y.W., Shin, T., Hyun, S.H., Park, J.K., Ki, M.R., Han, S.Y., Park, S.I., Lee, Langenmayer, M.C., Stehr, M., Holzinger, A., Kroner, C., and others, 2012.
J.H., Lee, E.M., and others, 2011a. Morphological abnormalities, impaired fetal Sequential targeting of CFTR by BAC vectors generates a novel pig model of
development and decrease in myostatin expression following somatic cell cystic fibrosis. Journal of Molecular Medicine 90, 597–608.
nuclear transfer in dogs. Molecular Reproduction and Development 78, 337– Kragh, P.M., Nielsen, A.L., Li, J., Du, Y., Lin, L., Schmidt, M., Bogh, I.B., Holm, I.E.,
346. Jakobsen, J.E., Johansen, and others, 2009. Hemizygous minipigs
Hong, S.G., Koo, O.J., Oh, H.J., Park, J.E., Kim, M., Kim, G.A., Park, E.J., Jang, G., Lee, B.C., produced by random gene insertion and handmade cloning express the
2011b. Post mortem re-cloning of a transgenic red fluorescent protein dog. Alzheimer’s disease-causing dominant mutation APPsw. Transgenic Research
Journal of Veterinary Science 12, 405–407. 18, 545–558.
Hori, T., Tsutsui, T., 2003. In vitro fertilisation of mature canine ova. Veterinary Kues, W.A., Sudheer, S., Herrmann, D., Carnwath, J.W., Havlicek, V., Besenfelder, U.,
Record 152, 688–690. Lehrach, H., Adjaye, J., Niemann, H., 2008. Genome-wide expression profiling
Hossein, M.S., Jeong, Y.W., Park, S.W., Kim, J.J., Lee, E., Ko, K.H., Hyuk, P., Hoon, S.S., reveals distinct clusters of transcriptional regulation during bovine
Kim, Y.W., Hyun, and others, 2009. Birth of Beagle dogs by somatic cell nuclear preimplantation development in vivo. Proceedings of the National Academy
transfer. Animal Reproduction Science 114, 404–414. of Sciences USA 105, 19768–19773.
Hu, J., Ma, X., Bao, J.C., Li, W., Cheng, D., Gao, Z., Lei, A., Yang, C., Wang, H., 2011. Kues, W.A., Herrmann, D., Barg-Kues, B., Meena Haridoss, S., Nowak-Imialek, M.,
Insulin–transferrin–selenium (ITS) improves maturation of porcine oocytes Buchholz, T., Streeck, M., Grebe, A., Grabundzija, I., Merkert, S., and others, 2012.
in vitro. Zygote 19, 191–197. Derivation and characterization of Sleeping Beauty transposon-mediated
Huang, B., Li, T., Alonso-Gonzalez, L., Gorre, R., Keatley, S., Green, A., Turner, P., porcine induced pluripotent stem cells. Stem Cells and Development. http://
Kallingappa, P.K., Verma, V., Oback, B., 2011. A virus-free poly-promoter vector dx.doi.org/10.1089/scd.2012.0382.
induces pluripotency in quiescent bovine cells under chemically defined Kuijk, E.W., Chuva de Sousa Lopes, S.M., Geijsen, N., Macklon, N., Roelen, B.A., 2011.
conditions of dual kinase inhibition. PLoS One 6, e24501. The different shades of mammalian pluripotent stem cells. Human
Hussein, S.M., Nagy, A.A., 2012. Progress made in the reprogramming field: New Reproduction Update 17, 254–271.
factors, new strategies and a new outlook. Current Opinion in Genetics and Lagutina, I., Lazzari, G., Duchi, R., Colleoni, S., Ponderato, N., Turini, P., Crotti, G.,
Development 22, 435–443. Galli, C., 2005. Somatic cell nuclear transfer in horses: Effect of oocyte
 V. Hall et al. / The Veterinary Journal 197 (2013) 128–142 141

morphology, embryo reconstruction method and donor cell type. Reproduction Oh, H.J., Park, J.E., Kim, M.J., Kim, G., Park, E.J., Lim, S.H., Kim, T.W., Cho, J., Jang, G.,
130, 559–567. Lee, B.C., 2012. Neuron-specific expression of the red fluorescence protein in
Lagutina, I., Lazzari, G., Duchi, R., Turini, P., Tessaro, I., Brunetti, D., Colleoni, S., cloned dogs. Reproduction Fertility and Development 24, 128.
Crotti, G., Galli, C., 2007. Comparative aspects of somatic cell nuclear transfer Oriol, J.G., Betteridge, K.J., Clarke, A.J., Sharom, F.J., 1993a. Mucin-like glycoproteins
with conventional and zona-free method in cattle, horse, pig and sheep. in the equine embryonic capsule. Molecular Reproduction and Development 34,
Theriogenology 67, 90–98. 255–265.
Lazzari, G., Wrenzycki, C., Herrmann, D., Duchi, R., Kruip, T., Niemann, H., Galli, C., Oriol, J.G., Sharom, F.J., Betteridge, K.J., 1993b. Developmentally regulated changes
2002. Cellular and molecular deviations in bovine in vitro-produced embryos in the glycoproteins of the equine embryonic capsule. Journal of Reproduction
are related to the large offspring syndrome. Biology of Reproduction 67, 767– and Fertility 99, 653–664.
775. Otoi, T., Murakami, M., Fujii, M., Tanaka, M., Ooka, A., Une, S., Suzuki, T., 2000.
Lee, B.C., Kim, M.K., Jang, G., Oh, H.J., Yuda, F., Kim, H.J., Hossein, M.S., Kim, J.J., Kang, Development of canine oocytes matured and fertilised in vitro. Veterinary
S.K., Schatten, G., Hwang, W.S., 2005. Dogs cloned from adult somatic cells. Record 146, 52–53.
Nature 436, 641. Park, J.E., Oh, H.J., Hong, S.G., Kang, J.T., Kim, M.J., Kim, D.Y., Ra, J.C., Goo, J., Lee, B.C.,
Levi, M., Maro, B., Shalgi, R., 2010. Fyn kinase is involved in cleavage furrow 2009. Cloning of cancer sniffing dog by somatic cell nuclear transfer.
ingression during meiosis and mitosis. Reproduction 140, 827–834. Reproduction Fertility and Development 21, 124–125.
Li, X., Zhou, S.G., Imreh, M.P., hrlund-Richter, L., Allen, W.R., 2006. Horse embryonic Park, J.E., Kim, M.K., Kang, J.T., Oh, H.J., Hong, S.G., Kim, D.Y., Jang, G., Lee, B.C., 2010.
stem cell lines from the proliferation of inner cell mass cells. Stem Cells and Growth and hematologic characteristics of cloned dogs derived from adult
Development 15, 523–531. somatic cell nuclear transfer. Cellular Reprogramming 12, 141–150.
Liu, Y., Ostrup, O., Li, J., Vajta, G., Kragh, P.M., Purup, S., Callesen, H., 2011. Cell colony Park, K.E., Johnson, C.M., Wang, X., Cabot, R.A., 2011. Differential developmental
formation induced by Xenopus egg extract as a marker for improvement of requirements for individual histone H3K9 methyltransferases in cleavage-stage
cloned blastocyst formation in the pig. Cellular Reprogramming 13, 521–526. porcine embryos. Reproduction, Fertility, and Development 23, 551–560.
Liu, Y., Ostrup, O., Li, J., Vajta, G., Lin, L., Kragh, P.M., Purup, S., Hyttel, P., Callesen, H., Parrish, J.J., Susko-Parrish, J.L., Leibfried-Rutledge, M.L., Critser, E.S., Eyestone, W.H.,
2012. Increased blastocyst formation of cloned porcine embryos produced with First, N.L., 1986. Bovine in vitro fertilization with frozen-thawed semen.
donor cells pre-treated with Xenopus egg extract and/or digitonin. Zygote 20, Theriogenology 25, 591–600.
61–66. Peat, J.R., Reik, W., 2012. Incomplete methylation reprogramming in SCNT embryos.
Lonergan, P., Fair, T., Corcoran, D., Evans, A.C., 2006. Effect of culture environment Nature Genetics 44, 965–966.
on gene expression and developmental characteristics in IVF-derived embryos. Petters, R.M., Wells, K.D., 1993. Culture of pig embryos. Journal of Reproduction and
Theriogenology 65, 137–152. Fertility Suppl. 48, 61–73.
Luo, J., Suhr, S.T., Chang, E.A., Wang, K., Ross, P.J., Nelson, L.L., Venta, P.J., Knott, J.G., Powell, A.M., Talbot, N.C., Wells, K.D., Kerr, D.E., Pursel, V.G., Wall, R.J., 2004. Cell
Cibelli, J.B., 2011. Generation of leukemia inhibitory factor and basic fibroblast donor influences success of producing cattle by somatic cell nuclear transfer.
growth factor-dependent induced pluripotent stem cells from canine adult Biology of Reproduction 71, 210–216.
somatic cells. Stem Cells and Development 20, 1669–1678. Prather, R.S., Barnes, F.L., Sims, M.M., Robl, J.M., Eyestone, W.H., First, N.L., 1987.
Luvoni, G.C., Chigioni, S., Allievi, E., Macis, D., 2005. Factors involved in vivo and Nuclear transplantation in the bovine embryo: Assessment of donor nuclei and
in vitro maturation of canine oocytes. Theriogenology 63, 41–59. recipient oocyte. Biology of Reproduction 37, 859–866.
Magnani, L., Cabot, R.A., 2007. Developmental arrest induced in cleavage stage Ralston, A., Rossant, J., 2008. Cdx2 acts downstream of cell polarization to cell-
porcine embryos following microinjection of mRNA encoding Brahma (Smarca autonomously promote trophectoderm fate in the early mouse embryo.
2), a chromatin remodeling protein. Molecular Reproduction and Development Developmental Biology 313, 614–629.
74, 1262–1267. Renner, S., Fehlings, C., Herbach, N., Hofmann, A., von Waldthausen, D.C., Kessler, B.,
Mahi, C.A., Yanagimachi, R., 1976. Maturation and sperm penetration of canine Ulrichs, K., Chodnevskaja, I., Moskalenko, V., Amselgruber, W., and others, 2010.
ovarian oocytes in vitro. Journal of Expimental Zoololy 196, 189–196. Glucose intolerance and reduced proliferation of pancreatic beta-cells in
Martin, G.R., 1981. Isolation of a pluripotent cell line from early mouse embryos transgenic pigs with impaired glucose-dependent insulinotropic polypeptide
cultured in medium conditioned by teratocarcinoma stem cells. Proceedings of function. Diabetes 59, 1228–1238.
the National Academy of Sciences USA 78, 7634–7638. Reynaud, K., Fontbonne, A., Marseloo, N., Viaris de Lesegno, C., Saint-Dizier, M.,
Maruotti, J., Munoz, M., Degrelle, S.A., Gomez, E., Louet, C., Monforte, C.D., de Chastant-Maillard, S., 2006. In vivo canine oocyte maturation, fertilization and
Longchamp, P.H., Brochard, V., Hue, I., Caamano, J.N., Jouneau, A., 2012. Efficient early embryogenesis: A review. Theriogenology 66, 1685–1693.
derivation of bovine embryonic stem cells needs more than active core Ribeiro, B.I., Love, L.B., Choi, Y.H., Hinrichs, K., 2008. Transport of equine ovaries for
pluripotency factors. Molecular Reproduction and Development 79, 461–477. assisted reproduction. Animal Reproduction Science 108, 171–179.
McPartlin, L.A., Suarez, S.S., Czaya, C.A., Hinrichs, K., Bedford-Guaus, S.J., 2009. Saint-Dizier, M., Renard, J.P., Chastant-Maillard, S., 2001. Induction of final
Hyperactivation of stallion sperm is required for successful in vitro fertilization maturation by sperm penetration in canine oocytes. Reproduction 121, 97–105.
of equine oocytes. Biology of Reproduction 81, 199–206. Saito, S., Ugai, H., Sawai, K., Yamamoto, Y., Minamihashi, A., Kurosaka, K., Kobayashi,
Memili, E., First, N.L., 1999. Control of gene expression at the onset of bovine Y., Murata, T., Obata, Y., Yokoyama, K., 2002. Isolation of embryonic stem-like
embryonic development. Biology of Reproduction 61, 1198–1207. cells from equine blastocysts and their differentiation in vitro. FEBS Letters 531,
Mikawa, T., Poh, A.M., Kelly, K.A., Ishii, Y., Reese, D.E., 2004. Induction and 389–396.
patterning of the primitive streak, an organizing center of gastrulation in the Saito, S., Sawai, K., Ugai, H., Moriyasu, S., Minamihashi, A., Yamamoto, Y., Hirayama,
amniote. Developmental Dynamics 229, 422–432. H., Kageyama, S., Pan, J., Murata, T. and others, 2003. Generation of
Mitalipova, M., Beyhan, Z., First, N.L., 2001. Pluripotency of bovine embryonic cell cloned calves and transgenic chimeric embryos from bovine embryonic
line derived from precompacting embryos. Cloning 3, 59–67. stem-like cells. Biochemical and Biophysical Research Communication 309,
Mortensen, C.J., Choi, Y.H., Ing, N.H., Kraemer, D.C., Vogelsang, M.M., Hinrichs, K., 104–113.
2010. Heat shock protein 70 gene expression in equine blastocysts after Schafer-Somi, S., Beceriklisoy, H.B., Budik, S., Kanca, H., Aksoy, O.A., Polat, B., Cetin,
exposure of oocytes to high temperatures in vitro or in vivo after exercise of Y., Ay, S.S., Aslan, S., 2008. Expression of genes in the canine pre-implantation
donor mares. Theriogenology 74, 374–383. uterus and embryo: Implications for an active role of the embryo before and
Mugnier, S., Dell’Aquila, M.E., Pelaez, J., Douet, C., Ambruosi, B., De Santis, T., during invasion. Reproduction in Domestic Animals (Zuchthygiene) 43, 656–
Lacalandra, G.M., Lebos, C., Sizaret, P.Y., Delaleu, B., and others, 2009. New 663.
insights into the mechanisms of fertilization: comparison of the fertilization Schmidt, M., Kragh, P.M., Li, J., Du, Y., Lin, L., Liu, Y., Bogh, I.B., Winther, K.D., Vajta, G.,
steps, composition, and structure of the zona pellucida between horses and Callesen, H., 2010. Pregnancies and piglets from large white sow recipients after
pigs. Biology of Reproduction 81, 856–870. two transfer methods of cloned and transgenic embryos of different pig breeds.
Munoz, M., Diez, C., Caamano, J.N., Jouneau, A., Hue, I., Gomez, E., 2008. Embryonic Theriogenology 74, 1233–1240.
stem cells in cattle. Reproduction in Domestic Animals 43, 32–37. Schneider, M.R., Adler, H., Braun, J., Kienzle, B., Wolf, E., Kolb, H.J., 2007. Canine
Nagy, K., Sung, H.K., Zhang, P., Laflamme, S., Vincent, P., Agha-Mohammadi, S., embryo-derived stem cells – Toward clinically relevant animal models for
Woltjen, K., Monetti, C., Michael, I.P., Smith, L.C., Nagy, A., 2011. Induced evaluating efficacy and safety of cell therapies. Stem Cells (Dayton, Ohio) 25,
pluripotent stem cell lines derived from equine fibroblasts. Stem Cell Reviews 7, 1850–1851.
693–702. Schurmann, A., Wells, D.N., Oback, B., 2006. Early zygotes are suitable recipients for
Nichols, J., Smith, A., 2009. Naive and primed pluripotent states. Cell Stem Cell 4, bovine somatic nuclear transfer and result in cloned offspring. Reproduction
487–492. 132, 839–848.
Oback, B., Wells, D.N., 2003. Cloning cattle. Cloning and Stem Cells 5, 243–256. Smith, S.L., Everts, R.E., Tian, X.C., Du, F., Sung, L.Y., Rodriguez-Zas, S.L., Jeong, B.S.,
Oback, B., Wiersema, A. T., Gaynor, P., Laible, G., Tucker, F. C., Oliver, J. E., Miller, A. L., Renard, J.P., Lewin, H.A., Yang, X., 2005. Global gene expression profiles reveal
Troskie, H. E., Wilson, K. L., Forsyth, J. T. and others, 2003. Cloned cattle derived significant nuclear reprogramming by the blastocyst stage after cloning.
from a novel zona-free embryo reconstruction system. Cloning Stem Cells 5, 3– Proceedings of the National Academy of Sciences USA 102, 17582–17587.
12. Smits, K., Goossens, K., Van Soom, A., Govaere, J., Hoogewijs, M., Peelman, L.J., 2011.
Oh, H.J., Kim, M.K., Jang, G., Kim, H.J., Hong, S.G., Park, J.E., Park, K., Park, C., Sohn, In vivo-derived horse blastocysts show transcriptional upregulation of
S.H., Kim, D.Y., Shin, N.S., Lee, B.C., 2008. Cloning endangered gray wolves (Canis developmentally important genes compared with in vitro-produced horse
lupus) from somatic cells collected postmortem. Theriogenology 70, 638–647. blastocysts. Reproduction Fertility Development 23, 364–375.
Oh, H.J., Park, J.E., Kim, M.J., Hong, S.G., Ra, J.C., Jo, J.Y., Kang, S.K., Jang, G., Lee, B.C., Songsasen, N., Wildt, D.E., 2007. Oocyte biology and challenges in developing
2011. Recloned dogs derived from adipose stem cells of a transgenic cloned in vitro maturation systems in the domestic dog. Animal Reproduction Science
beagle. Theriogenology 75, 1221–1231. 98, 2–22.
142 V. Hall et al. / The Veterinary Journal 197 (2013) 128–142

Starkey, M.P., Scase, T.J., Mellersh, C.S., Murphy, S., 2005. Dogs really are man’s best polyspermy after in vitro fertilization. Molecular Reproduction and
friend–canine genomics has applications in veterinary and human medicine! Development 49, 308–316.
Brief Funct Genomic Proteomic 4, 112–128. Wang, L., Duan, E., Sung, L.Y., Jeong, B.S., Yang, X., Tian, X.C., 2005. Generation and
Staunstrup, N.H., Madsen, J., Primo, M.N., Li, J., Liu, Y., Kragh, P.M., Li, R., Schmidt, M., characterization of pluripotent stem cells from cloned bovine embryos. Biology
Purup, S., Dagnaes-Hansen, F., and others, 2012. Development of transgenic of Reproduction 73, 149–155.
cloned pig models of skin inflammation by DNA transposon-directed ectopic Wells, D.N., 2005. Animal cloning: Problems and prospects. Reviews in Science and
expression of human beta1 and alpha2 integrin. PloS one 7, e36658. Technology 24, 251–264.
Stice, S.L., Strelchenko, N.S., Keefer, C.L., Matthews, L., 1996. Pluripotent bovine Wells, D.N., Misica, P.M., Tervit, H.R., Vivanco, W.H., 1998. Adult somatic cell nuclear
embryonic cell lines direct embryonic development following nuclear transfer. transfer is used to preserve the last surviving cow of the Enderby Island cattle
Biology of Reproduction 54, 100–110. breed. Reproduction, Fertility, and Development 10, 369–378.
Sun, Q.Y., Wu, G.M., Lai, L., Park, K.W., Cabot, R., Cheong, H.T., Day, B.N., Prather, R.S., Welsh, M.J., Rogers, C.S., Stoltz, D.A., Meyerholz, D.K., Prather, R.S., 2009.
Schatten, H., 2001. Translocation of active mitochondria during pig oocyte Development of a porcine model of cystic fibrosis. Transactions of the
maturation, fertilization and early embryo development in vitro. Reproduction American Clinical and Climatological Association 120, 149–162.
122, 155–163. West, F.D., Terlouw, S.L., Kwon, D.J., Mumaw, J.L., Dhara, S.K., Hasneen, K.,
Suzuki, S., Iwamoto, M., Saito, Y., Fuchimoto, D., Sembon, S., Suzuki, M., Mikawa, S., Dobrinsky, J.R., Stice, S.L., 2010. Porcine induced pluripotent stem cells
Hashimoto, M., Aoki, Y., Najima, Y., and others, 2012. Il2rg gene-targeted severe produce chimeric offspring. Stem Cells and Development 19, 1211–1220.
combined immunodeficiency pigs. Cell Stem Cell 10, 753–758. West, F.D., Uhl, E.W., Liu, Y., Stowe, H., Lu, Y., Yu, P., Gallegos-Cardenas, A., Pratt, S.L.,
Talbot, N.C., Powell, A.M., Rexroad Jr., C.E., 1995. In vitro pluripotency of epiblasts Stice, S.L., 2011. Brief report: Chimeric pigs produced from induced pluripotent
derived from bovine blastocysts. Molecular Reproduction and Development 42, stem cells demonstrate germline transmission and no evidence of tumor
35–52. formation in young pigs. Stem Cells 29, 1640–1643.
Takahashi, K., Yamanaka, S., 2006. Induction of pluripotent stem cells from mouse Whitworth, D.J., Ovchinnikov, D.A., Wolvetang, E.J., 2012. Generation and
embryonic and adult fibroblast cultures by defined factors. Cell 126, 663–676. characterization of LIF-dependent canine induced pluripotent stem cells from
Telugu, B.P., Ezashi, T., Sinha, S., Alexenko, A.P., Spate, L., Prather, R.S., Roberts, R.M., adult dermal fibroblasts. Stem Cells and Development 21, 2288–2297.
2011. Leukemia inhibitory factor (LIF)-dependent, pluripotent stem cells Wilcox, J.T., Semple, E., Gartley, C., Brisson, B.A., Perrault, S.D., Villagomez, D.A.,
established from inner cell mass of porcine embryos. The Journal of Biological Tayade, C., Becker, S., Lanza, R., Betts, D.H., 2009. Characterization of canine
Chemistry 286, 28948–28953. embryonic stem cell lines derived from different niche microenvironments.
Tesar, P.J., Chenoweth, J.G., Brook, F.A., Davies, T.J., Evans, E.P., Mack, D.L., Gardner, Stem Cells and Development 18, 1167–1178.
R.L., McKay, R.D., 2007. New cell lines from mouse epiblast share defining Wilcox, J.T., Lai, J.K., Semple, E., Brisson, B.A., Gartley, C., Armstrong, J.N., Betts, D.H.,
features with human embryonic stem cells. Nature 448, 196–199. 2011. Synaptically-competent neurons derived from canine embryonic stem
Tesoriero, J.V., 1981. Early ultrastructural changes of developing oocytes in the dog. cells by lineage selection with EGF and Noggin. PLoS One 6, e19768.
Journal of Morphology 168, 171–179. Willadsen, S.M., 1986. Nuclear transplantation in sheep embryos. Nature 320, 63–
Thomassen, R., Farstad, W., 2009. Artificial insemination in canids: A useful tool in 65.
breeding and conservation. Theriogenology 71, 190–199. Wilmut, I., Schnieke, A.E., McWhir, J., Kind, A.J., Campbell, K.H., 1997. Viable
Thomson, J.A., Itskovitz-Eldor, J., Shapiro, S.S., Waknitz, M.A., Swiergiel, J.J., Marshall, offspring derived from fetal and adult mammalian cells. Nature 385, 810–813.
V.S., Jones, J.M., 1998. Embryonic stem cell lines derived from human Wolf, X.A., Serup, P., Hyttel, P., 2011. Three-dimensional localisation of NANOG,
blastocysts. Science 282, 1145–1147. POU5F1, and E-CADHERIN in porcine pre- and peri-implantation embryos.
Tremoleda, J.L., Stout, T.A., Lagutina, I., Lazzari, G., Bevers, M.M., Colenbrander, B., Developmental Dynamics: An Official Publication of the American Association
Galli, C., 2003. Effects of in vitro production on horse embryo morphology, of Anatomists 240, 204–210.
cytoskeletal characteristics, and blastocyst capsule formation. Biology of Woods, G.L., White, K.L., Vanderwall, D.K., Li, G.P., Aston, K.I., Bunch, T.D., Meerdo,
Reproduction 69, 1895–1906. L.N., Pate, B.J., 2003. A mule cloned from fetal cells by nuclear transfer. Science
Vaags, A.K., Rosic-Kablar, S., Gartley, C.J., Zheng, Y.Z., Chesney, A., Villagomez, D.A., 301, 1063.
Kruth, S.A., Hough, M.R., 2009. Derivation and characterization of canine Wu, Z., Chen, J., Ren, J., Bao, L., Liao, J., Cui, C., Rao, L., Li, H., Gu, Y., Dai, H., and others,
embryonic stem cell lines with in vitro and in vivo differentiation potential. 2009. Generation of pig induced pluripotent stem cells with a drug-inducible
Stem Cells 27, 329–340. system. Journal of Molecular Cell Biology 1, 46–54.
Vajta, G., Callesen, H., 2012. Establishment of an efficient somatic cell nuclear Wu, G., Gentile, L., Fuchikami, T., Sutter, J., Psathaki, K., Esteves, T.C., Arauzo-Bravo,
transfer system for production of transgenic pigs. Theriogenology 77, 1263– M.J., Ortmeier, C., Verberk, G., Abe, K., Scholer, H.R., 2010. Initiation of
1274. trophectoderm lineage specification in mouse embryos is independent of
van Wagtendonk-de Leeuw, A.M., Mullaart, E., de Roos, A.P., Merton, J.S., den Daas, Cdx2. Development 137, 4159–4169.
J.H., Kemp, B., de Ruigh, L., 2000. Effects of different reproduction techniques: AI Yamada, S., Shimazu, Y., Kawaji, H., Nakazawa, M., Naito, K., Toyoda, Y., 1992.
MOET or IVP, on health and welfare of bovine offspring. Theriogenology 53, Maturation, fertilization, and development of dog oocytes in vitro. Biology of
575–597. Reproduction 46, 853–858.
Varner, G., 1999. Should you clone your dog? An animal rights perspective on Yamada, S., Shimazu, Y., Kawano, Y., Nakazawa, M., Naito, K., Toyoda, Y., 1993. In
somacloning. Animal Welfare 8, 407–420. vitro maturation and fertilization of preovulatory dog oocytes. Journal of
Vejlsted, M., Du, Y., Vajta, G., Maddox-Hyttel, P., 2006. Post-hatching development Reproduction and Fertility Suppl. 47, 227–229.
of the porcine and bovine embryo – Defining criteria for expected development Ying, Q.L., Wray, J., Nichols, J., Batlle-Morera, L., Doble, B., Woodgett, J., Cohen, P.,
in vivo and in vitro. Theriogenology 65, 153–165. Smith, A., 2008. The ground state of embryonic stem cell self-renewal. Nature
Vignon, X., Chesne, P., Le Bourhis, D., Flechon, J.E., Heyman, Y., Renard, J.P., 1998. 453, 519–523.
Developmental potential of bovine embryos reconstructed from enucleated Yoshioka, K., Suzuki, C., Onishi, A., 2008. Defined system for in vitro production of
matured oocytes fused with cultured somatic cells. Comptes Rendus de porcine embryos using a single basic medium. The Journal of Reproduction and
l’Académie des Sciences – Series III 321, 735–745. Development 54, 208–213.
Wang, W.H., Abeydeera, L.R., Prather, R.S., Day, B.N., 1998. Morphologic comparison Young, L.E., Sinclair, K.D., Wilmut, I., 1998. Large offspring syndrome in cattle and
of ovulated and in vitro-matured porcine oocytes, with particular reference to sheep. Reviews of Reproduction 3, 155–163.