




































In ternationa l
Scholars
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African Journal of Pig Farming ISSN 2375-0731 Vol. 7 (1), pp. 001-004, January, 2019. Available online at 
www.internationalscholarsjournals.org © International Scholars Journals 

 

Author(s) retain the copyright of this article. 

 

Review 
 

Review of somatic cell nuclear transfer in pig 

 
Suchitra Muenthaisong1,2, Andras Dinnyes1,2,3 and Tshimangadzo Lucky Nedambale4,5,6*

 
 

1
Molecular Animal Biotechnology Laboratory, Szent Istvan University, H-2100 Gödöllö, Hungary. 

2
BioTalentum Ltd, H-2100 Gödöllö, Hungary. 

3
Dept. of Farm Animal Health, Faculty of Veterinary Medicine, Utrecht University, The Netherlands. 

4
Agricultural Research Council (ARC), Animal Production Institute, Germplasm Conservation and Reproductive 

Biotechnologies, Irene, 0062, South Africa.  
5
Tshwane University of Technology, Department of Animal Sciences, Private Bag X680, Pretoria 0001, 

South Africa. 
6
University of the Free State, Department of Animal, Wildlife and Grassland Sciences, P.O. 

Box 339, Bloemfontein, South Africa. 
 

Accepted 30 September, 2018 
 
It is now more than 8 years, since the first cloned pig from nuclear transfer was reported. Success of somatic 
cell nuclear transfer (SCNT) in pig is still low compared to that in bovine. Embryonic and neonatal abnormalities 
of cloned piglets are probably a result of incorrect or incomplete reprogramming of the transferred donor cell 
nuclei. Recently, technical refinements have resulted in a considerable increase of SCNT efficiency. The current 
level of efficiency is already sufficient for special purposes with high scientific and commercial impact including 
xenotransplantation, generation of human disease models and for biomedical studies. In this paper, we review 
current pig cloning methodologies for the technical and potential applications. 
 
Key words: Pig, somatic cell, nuclear transfer, oocyte and embryo. 

 
INTRODUCTION 

 
To date, cloned offspring by somatic cell nuclear transfer 
(Figure 1) have been successfully produced in a variety 
of mammals (Wilmut et al., 1997; Wakayama et al., 1998; 
Kato et al., 1998; Onishi et al., 2000). However, the 
success rate remains low and many important factors 
remain unclear especially in pig compared to other 
species (Wu et al., 2001). Usually, pig oocytes and 
embryos are very sensitive to stressors, physical change 
and chemical factors for nuclear transfer technology. The 
pig oocytes contain many lipids inside the cytoplasm 
which explain their sensitivity to environmental stressors. 
It seems that lipids in pig oocytes affect the development 
of embryos following somatic nuclear transfer technique 
(Nagashima et al., 1995). Several reports have indicated 
that the oocytes activation, embryo culture and embryo 
transfer and early embryo developmental stage seem to  
 
 
 
*Corresponding author. E-mail: lucky@arc.agric.za. Tel: +2712 
672 9200. Fax: +2712 665 1604. 

 
 
 
 

 
be the major problems of SCNT in pigs. However, there is 
a considerable improvement on the efficiency of SCNT in 
pigs and application such as cloned transgenic pigs to be 
used for xenotransplantation, are under intensive 
research (Dai et al., 2002; Lai et al., 2002b; Kolber-
Simonds et al., 2004).  

The objective of this review was to provide an overview 
of the somatic cell nuclear transfer in pig and summarize 
recent improvement on the efficiency of pig SCNT 
technology. 

 
IN VITRO MATURATION (IVM) OF PIG OOCYTE 

 
In SCNT technique, the oocytes are needed to use for 
recipient cytoplasm. Most of the recipient oocytes are 
matured IN VITRO or derived from IN VIVO. The first two 
reports using IN VIVO matured pig oocytes for SCNT 
reported successfully application of SCNT and they 
produced cloned piglet (Onishi et al., 2000; Polejaeva et 



  
 
 

 

Egg donor Nuclei donor  
 
 

 
Propagate the  
single donor cell  

 
 
 

 
Remove the egg  
nucleus  

 
 
 

 
Inject the nuclei donor  

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

Oocyte were  
electrical activated  

 
 
 
 

 
Transferred reconstructed  
oocyte into surrogate mother 

 
 
 

 
Surrogate mother 

 
 

 
Cloned pigs 

 
Figure 1. Somatic cell nuclear transfer (SCNT) in pig. 

 

 

al., 2000). As a result, cloned piglet was also born using 
IN VITRO matured oocytes from the ovaries derived from 

slaughter house (Betthauser et al., 2000).  
The North Carolina State University (NCSU) 23 and 

tissue culture medium (TCM) 199 have been 

 
 

 

compared for maturation of pig oocytes and no 
differences were found among them on supporting the 
development of cloned pig embryos (Hyun et al., 2003a). 
Several researchers are interested on improving oocyte 
and embryo developmental capacity by modify the 



 
 
 

 

culture media and culture conditions to enhance the 
successful development of cloned embryos IN VITRO. 
Generally, the basic medium for pig oocytes is NCSU 23 
or NCSU-37 (Petters and Wells, 1993).  

For IN VITRO maturation, pig follicular fluid (pFF) was 
used to supplement in the IVM medium. The pFF is 
usually added to maturation media of pig oocytes to 
protecting oocytes from oxidative stress through a higher 
level of radical scavenging activity, resulting in the 
enhancement of cytoplasmic maturation for development 
competence after fertilization. The maturation medium 
that supplemented with pFF can enhanced development 
competence of pig maturation. The factors within the 
follicles may play an important role to support the oocytes 
during maturation (Schoevers et al., 2003; Algriany et al., 
2004). However, pFF can be contaminated from 
numerous undefined factors with viral pathogens (Kim 
and Dubovi, 2003).  

The addition of hormones, growth factors, vitamins, 
energy substrates, inorganic compound, cytokines and 
follicular fluid influences considerably maturation and 
subsequent development of pig oocytes (Abeydeera, 
2002). There is a report that epidermal growth factor 
(EGF) plays an important role in nuclear and cytoplasmic 
maturation of pig oocytes (Grupen et al., 1997; 
Abeydeera et al., 2000). Moreover, exposure of cumulus-
oocyte complexes (COCs) to dibutyryl cAMP (dbcAMP) 
for maturation increased developmental competent of pig 
oocytes (Funahashi et al., 1997). Recently, defined 
systems for IN VITRO production of pig embryos using a 
single basic medium were reported for blastocyst 
production from chemically defined pig gamete medium 
(PGM) and pig zygote medium (PZM). The media based 
on the composition of pig oviductal fluid and appropriately 
supplemented for specific stage of embryo development 
thus can be used for IN VITRO maturation, fertilization and 
culture in pig (Yoshioka et al., 2008). 
 

 

SOMATIC CELL NUCLEAR TRANSFER 

 

It is a process known as nuclear transfer (NT) in which 
the nucleus of a donor cell is transferred into an oocyte. 
The first pig cloned by SCNT was reported by Prather et 
al. (1989). They used pronuclei exchange zygote and 
blastomere as a donor cell. Recipient cytoplasm derived 
from IN VIVO maturation was used and activated the 
reconstructed oocytes with electric before transfer to the 
oviduct for IN VIVO embryo culture. Blastocyst embryos 
were transferred to the recipient for develop into term. 
They received seven live pigs born from pronuclei 
exchange zygote and one live pig born from blastomere 
at 4-cell stage. Moreover, successful cloned pig by the 
use of somatic cell nuclear transfer technique has been 
reported by Polejaeva et al. (2000). They used granulosa 
cells as a donor cell with two-stage nuclear transfer 

 
 
 
 

 

technique that donor cells were fused to enucleated 
oocytes in the first stage, and the pronucleus-like  
structures formed were then subsequently 
transplantedinto IN VIVO-produced, enucleated zygotes. 
However, the successful production of cloned pig from a 
single-step nuclear transfer and the method is still wildly 
used in several laboratories to produce the SCNT pig 
(Betthauser et al., 2000; Onishi et al., 2000; Park et al., 
2001; De Sousa et al., 2002; Yin et al., 2002).  

Several researchers also reported the successful 
production of pig cloned with different donor cell types 
(Onishi et al., 2000; Betthauser et al., 2000; Bondioli et 
al., 2001; Park et al., 2002) and also they attempt to 
modify donor cells to improve the efficiency of nuclear 
transfer. The donor cell in SCNT can be prepared as 
synchronized (Onishi et al., 2000; Polejaeva et al., 2000) 
and non-synchronised donor cells (Betthauser et al., 
2000; Bondioli et al., 2001). There success the nuclear 
transfer with both of IN VIVO-matured oocytes (Onishi et al., 
2000; Polejaeva et al., 2000) and IN VITRO-matured 
oocytes (Betthauser et al., 2000). Many strategies to 
introduce the donor cell into cytoplasm of the recipient 
cytoplasm such as transferred by electrofusion 
(Polejaeva et al., 2000), by piezo-microinjection of 
isolated donor nuclei (Onishi et al., 2000), and also by 
whole cell injection (Lee et al., 2003b). Recently, the 
zona-free technique become of interest with zona 
removal before or after enucleation for fusion. (Booth et 
al., 2001; Kragh et al., 2004; Du et al., 2005). 
 

 

ARTIFICIAL ACTIVATION 

 

In an effort to establish the cloned embryos, activation is 
the part of important step to initial reconstructs oocytes 
and donor cells. Since sperm-mediated activation is 
absent in SCNT, an artificial activation is needed to 
initiate embryo development. Mostly in mammals, 
oocytes are arrested at metaphase II (MII) stage after 
ovulation and complete meiosis after fertilization. The MII 
arrest is characterized by MPF activity (Nurse, 1990). 
Since the sperm-induce the release from meiotic arrest 
by a signal transduction pathway, calcium, M-phase 
promoting factor (MPF) and its stabilizing molecules play 
a role during oocyte activation by inducing a transient 
increase in the intracellular free calcium concentration by 
artificial means.  

For IN VITRO culture of pig embryos, oocytes are matured 
for around 42-44 h after the start of IVM, thus on this 
stage, a calcium transient is able to stimulate 
development (Machaty and Prather, 1998). There are 
many activation protocols to stimulate the oocyte such as 
electrical, chemical or mechanical activation. 
Simultaneous fusion/activation has been used for the 
generation of cloned pigs with targeted disruption of one 
or both alleles of the α-1,3-galactosyltransferase gene 



 
 
 

 

(Dai et al., 2002; Lai et al., 2002a; Kolber-Simonds et al., 
2004). The transgenic cloned pig expressing the green 
fluorescent protein were produced by triggering fusion 
and activation at the same time with an electrical stimulus 
(Park et al., 2001; Lai et al., 2002; Hyun et al., 2003b; 
Lee et al., 2005). Usually, high voltage direct current (DC) 
pulse is applied to generate an influx of extracellular 
calcium (Machaty and Prather, 1998).  

In the absence of extracellular calcium ions, the DC 
pulse induces membrane fusion only. In this case, high 
MPF levels in the oocyte cytoplasm cause to nuclear 
envelope breakdown and premature chromosome 
condensation. In the case of chemical activation, the 
oocytes were exposed to the substrates such as 
cytochalasin B (Li et al., 2000; Park et al., 2002; Hoshino 
et al., 2005) to prevent the extrusion of the second polar 
body, cycloheximide (Lee et al., 2003a) or 6-
dimethylaminopurine (DMAP; de la Fuente and King, 
1998; Hölker et al., 2005) to inhibit the protein kinase 
after an induced calcium transient to promote activation 
or ionomycin (Betthauser et al., 2000; Boquest et al., 
2002) that it forms a complex with calcium ions and 
transports through the plasma membrane and it can also 
stimulate calcium and induce a calcium influx similar 
electrical activation (Morgan and Jacob, 1994).  

Similarly, delayed activation was effective in supporting 
term development after demecolcine assisted enucleating 
(Yin et al., 2002; Kawakami et al., 2003) or mechanical 
activation when the donor nuclei were microinjected 
directly into the recipient cytoplasm (Onishi et al., 2000; 
Watanabe et al., 2005; Takeda et al., 2006). It has been 
suggested that nuclear reprogramming is more 
successful if chromosomes of the nuclear donor cell are 
exposed to the recipient oocyte cytoplasm for an 
extended period of time (Miyoshi et al. 2000; Tani et al. 
2001; Shin et al. 2002). Furthermore, development after 
nuclear transfer may be superior under delayed activation 
conditions compared to simultaneous fusion/activation 
(Yin et al., 2003). All the activation protocols mentioned 
above stimulate embryonic development by triggering a 
single calcium transient. However, the availability of 
current methods to induce repetitive calcium transient in 
pig oocyte is limited. 
 

 

EMBRYO CULTURE AND TRANSFER 

 

In order to establish pregnancies, the pig embryos were 
transfer to the recipient in early embryo development 
stage between one-cell to eight-cell stage with a many 
number of embryo. Most of embryo numbers were 
transferred more than 100 embryos to one recipient to 
avoid the negative effects of the IN VITRO system that 
seems to be effect to pig embryos more than other 
species. However, the pregnancy rate remains low and 
the fetus is almost lost during gestation (Chae et al., 

  
  

 
 

 

2006).  
Numerous studies aimed to improve the culture 

medium for preimplantation of embryo that can produce 
the cloned pig after transfer to the recipient. The result is 
considered to better understanding of pig embryonic 
development. The culture medium for pig embryo culture 
is NCSU-23 (Onishi et al., 2000; Polejaeva et al., 2000), 
NCSU-37 (Kikuchi et al., 2002) and Pig Zygote Medium 
(PZM)-3 (Yoshioka et al., 2002; Im et al., 2004). Recently, 
the result found that PZM-3 was superior in order to 
support the development of pig embryos, especially the 
embryos derived from SCNT. It seems that the PZM-3 
could better improve the efficiency of pig embryo to 
develop to preimplantation stage compared to NCSU-23 
(Im et al., 2004). Moreover, there is a report to study 
effect of bovine serum albumin (BSA) and polyvinyl 
alcohol (PVA) supplementation in NCSU-23. They found 
that both of them could enhance the production of pig 
embryo (Roh and Hwang, 2002).  

Regarding to culture condition under temperature and 
gases control incubator, the pig embryos cultured in an 

atmosphere with 5% CO2 in air showed the 
developmental rates and total cell numbers of blastocysts 

higher than embryos cultured under 5% CO2, 5% O2 and 

90% N2 (Machaty et al., 1998). Recently, there are 
reports showing that low oxygen concentration (five-
seven %) can enhance the pig embryo development from 
IN VITRO culture (Im et al., 2004; Sage et al., 2005). 
However, there were no differences developing in embryo 
development, total cell number and apoptosis incident 
under low and high oxygen concentration from IN VITRO 

fertilization and parthenogenetic pig embryos (Ock et al., 
2005).  

In addition, the modify culture conditions for cloned pig 
embryos by supplementation of various growth factors 
such as platelet activating factor (Kidson et al., 2004), 
EGF (Lee et al., 2004) showed significant increased of 
blastocyst rate and total cell number in pig SCNT 
embryos. Moreover, drop and Well of the Well (WOW) 
(Vajta et al., 2000) including the drop culture technique 
are modified culture systems that seem to be beneficial to 
establish the pig SCNT embryos (Taka et al., 2005). 
Regarding the attempts to modify the culture system for IN 

VITRO system, the ideal way used for cloned embryos in 

future experiment and in the physical environment may 
also have profound effect to IN VITRO development. 
 

 

PREGNANCY AND FARROWING 

 

Although, the cloned pigs were established from IN VITRO 
production, the overall efficiency is still very low in pig 
compared to cattle, sheep and mouse. Failures of SCNT 
pregnancy are associated with placental abnormalities, 
such as placentomegaly, reduced vascularisation, 
hypoplasia of trophoblastic epithelium, and altered 



 
 

 

 

basement membrane (Hill et al., 2000). Pregnancy has 
already been achieved with non-surgical transfer of IN 
VITRO-produced pig embryos (Suzuki et al., 2004). These 

may be due to intrinsic differences between pigs and 
these other species, or to differences in protocols for 
embryo production used. Furthermore, the larger litter 
size in pigs may also limit fetal growth to a greater extent 
than in cattle and sheep (Young et al., 1998). Especially 
that the number of pig embryos transferred to the 
recipient is very high than in another species (Koo et al., 
2004). Mostly pig embryos were transferred at the one-
cell; two-four-cell or four-eight-cell stages and 50-150 
embryos were transferred to one recipient. In pigs, there 
is a need for a signal of three or more embryos to 
maintain pregnancy (King et al., 2002). In case, of SCNT, 
this signal is weak, there are reports on the use of 
enhance and maintain the signal of pregnancy (Polejaeva 
et al., 2000; Lai et al., 2002b) or in vitro embryos by 
mating of recipient (Onishi et al., 2000; Lai et al., 2002b; 
Pan et al., 2006) or by hormone injection after embryo 
transfer (Walker et al., 2002; Harrison et al., 2004; Pan et 
al., 2006) that were successfully used in producing 
cloned pigs. 
 

 

APPLICATION OF PIG SOMATIC CELL NUCLEAR 
TRANSFER (SCNT) 

 

The mainly purpose of pig SCNT is related on the 
biomedical applications to human and also for 
xenotransplantation to replace organs or tissues by using 
the pig as an animal models for human diseases to 
therapy study because pig has a relatively similar organ 
size, anatomy, and physiology to that of human. 
Furthermore, their genome is much closer to human 
compared with mouse. The gene expression pattern of 
pigs is more closely similar to those of a human 
(Forsberg, 2005). However, there is a need to improve 
and increase the efficiency of pig SCNT and develop 
cloned pig with genetic modification that the organs will 
not cause an immunological response and destroy 
transplanted tissue when transferred to human. In 
addition, initial development and validation of the lab 
procedures, the availability of reliable and efficient 
methods for producing viable pig cloned following nuclear 
transfer technique still needs to be accomplished. 
 

 

ACKNOWLEDGEMENTS 

 

The authors thank the Hungarian-South African Bilateral 
Agreement Project (NRF-DST, TET OMFB-00302/2008), 
ARC-NRF-RT21000 and EU FP6 (MEDRAT-LSHG-CT-
2005-518240 and MRTN-CT-2006-035468) EU FP7 
(AniStem, PIAP-GA-2011-286264) projects for supporting 
the work resulting in this review article. 

 

 

 

 

 

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