1. 1 23
In Vitro Cellular & Developmental
Biology - Animal
ISSN 1071-2690
In Vitro Cell.Dev.Biol.-Animal
DOI 10.1007/s11626-016-0071-8
Production of hand-made cloned buffalo
(Bubalus bubalis) embryos from non-viable
somatic cells
E. K. A. Duah, S. K. Mohapatra,
T. J. Sood, A. Sandhu, S. K. Singla,
M. S. Chauhan, R. S. Manik & P. Palta

2. 1 23
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3. Production of hand-made cloned buffalo (Bubalus bubalis)
embryos from non-viable somatic cells
E. K. A. Duah1
& S. K. Mohapatra1
& T. J. Sood1
& A. Sandhu1
& S. K. Singla1
&
M. S. Chauhan1
& R. S. Manik1
& P. Palta1
Received: 25 April 2016 /Accepted: 3 July 2016 / Editor: Tetsuji Okamoto
# The Society for In Vitro Biology 2016
Abstract Use of non-viable somatic cells for hand-made
cloning (HMC) can enable production of cloned animals from
tissues obtained from elite or endangered dead animals.
Buffalo skin fibroblast cells were rendered non-viable by heat
treatment and used for HMC. Although fusion (93.6 ± 1.72 vs
67.1 ± 2.83%) and cleavage (90.3 ± 1.79 vs 65.8 ± 1.56%) rate
was lower (P < 0.001) than that for controls, blastocysts could
be successfully produced. However, blastocyst rate
(34.1 ± 2.43 vs 6.9 ± 2.18%, P < 0.001) and total cell number
of blastocysts (TCN, 221.3 ± 25.14 vs 151.1 ± 21.69,
P < 0.05) were lower and apoptotic index (4.8 ± 1.06 vs
10.9 ± 1.21) was higher (P < 0.001) than that of controls. In
another experiment, ear tissue of slaughterhouse buffaloes
was preserved in mustard oil at room temperature for 48 h
following which somatic cells were harvested by enzymatic
digestion and used for HMC. Although fusion (96.8 ± 1.48 vs
84.2 ± 3.19%), cleavage (89.6 ± 3.59 vs 77.2 ± 3.99%), and
blastocyst rate (36.9 ± 7.45 vs 13.1 ± 6.87%) were lower
(P < 0.01), TCN (223.0 ± 27.89 vs 213.3 ± 28.21) and apo-
ptotic index (3.97 ± 0.67 vs 5.22 ± 0.51) of blastocysts were
similar to those of controls. In conclusion, HMC can be suc-
cessfully used for production of blastocysts from non-viable
cells and from cells obtained from freshly slaughtered buffa-
loes. This can pave the way for the restoration of farm or wild
animals by HMC if somatic cells could be obtained within a
few hours after their death.
Keywords Cloning . SCNT . Nuclear transfer . Cloned
embryos
Introduction
Numerous cell types obtained from a multitude of tissue types
have been successfully used for somatic cell nuclear transfer
(SCNT). However, their viability has not been found to be a
critical requirement since live offsprings have been obtained
from SCNT embryos produced from heat-treated non-viable
sheep cells (Loi et al. 2002), cells obtained from mice bodies
frozen at −20°C for 16 yr (Wakayama et al. 2008) and testic-
ular cells taken from a dead bovine bull and frozen without
cryoprotectant in a −80°C freezer for 10 yr (Hoshino et al.
2009). The unprecedented decline of biodiversity worldwide
has prompted the collection and storage of biological material
from elite farm animals and seriously threatened animals for
their restoration and multiplication in the future. Recent suc-
cesses in SCNT have opened up new opportunities to restore
dead (Selokar et al. 2014), endangered, or even extinct mam-
malian species (Folch et al. 2009).
* P. Palta
prabhatpalta@yahoo.com
E. K. A. Duah
nkdekad@gmail.com
S. K. Mohapatra
drsushilmohapatra@gmail.com
T. J. Sood
tanushrijerath@gmail.com
A. Sandhu
anjitsandhu26@gmail.com
S. K. Singla
singlasuresh@yahoo.com
M. S. Chauhan
chauhanabtc@gmail.com
R. S. Manik
manik_rs@rediffmail.com
1
Embryo Biotechnology Laboratory, Animal Biotechnology Centre,
National Dairy Research Institute, Karnal 132001, India
In Vitro Cell.Dev.Biol.—Animal
DOI 10.1007/s11626-016-0071-8
Author's personal copy

4. Following the first successful use of somatic cells as nucle-
ar donors for SCNT in sheep, which led to the birth of BDolly^
(Wilmut et al. 1997), direct injection of donor nucleus into the
cytoplast was carried out successfully in several other species
such as cattle (Trounson et al. 1998), pig (Onishi et al. 2000),
horse (Choi et al. 2002), and buffalo (Shi et al. 2007). Vajta
et al. (2005) reported that more than 99% of scientific publi-
cations dealing with SCNT that had been published by 2005,
referred to micromanipulation-based enucleation and nuclear
transfer either by sub-zonal or intracytoplasmic injection of
donor cell or nucleus. Therefore, the SCNT was confined to
a few laboratories that could afford the expensive microma-
nipulators and had the skilled manpower to operate them.
Successful use of non-viable cells, obtained from tissues of
dead animals, offers a remarkable opportunity for restoration
of valuable animals through SCNT. However, till date, in all
the studies in which non-viable cells were used, SCNT was
carried out by conventional micromanipulation or whole cell
intracytoplasmic injection into an enucleated oocyte. These
techniques do not require the somatic cells to possess intact
plasma membrane capable of being electrofused with the oo-
cyte membrane since the nuclear material is injected directly
into the zona pellucida-enclosed oocyte. A major break-
through in the simplification and wider use of SCNT came
following the development of hand-made cloning (HMC) by
Vajta et al. (2001). This technique did not require microma-
nipulators because the manipulations required for both enu-
cleation and nucleus transfer were performed by hand. HMC
involves manual bisection of zona-free oocytes, selection of
cytoplasts by staining and the simultaneous fusion of the so-
matic cell with two cytoplasts to produce a cloned embryo. It
is not only much simpler, considerably less expensive and
rapid, but also the production efficiency is high and embryo
quality, in terms of pregnancy rates and live births, is not
compromised (Vajta et al. 2005). Therefore, HMC is nowa-
days preferred over the micromanipulation-based SCNT
(Vajta 2007). Presence of an intact plasma membrane in the
somatic cell, capable of being electrofused with the oocyte
membrane, is essential for HMC. To our knowledge, there is
no report till date on the use of non-viable cells as nuclear
donor for production of cloned embryos by HMC.
Therefore, we explored the possibility of producing cloned
buffalo embryos through HMC using non-viable cells and
somatic cells obtained from freshly slaughtered buffaloes.
Materials and methods
All the chemicals and media were purchased from
Sigma Chemical Co. (St. Louis, MO), the disposable
plasticware was from Nunc (Roskilde, Denmark) and
the media were from GIBCO (Grand Island, NY) unless
otherwise mentioned. Fetal bovine serum (FBS) was
obtained from Hyclone (Logan, UT). Ear skin fibroblast
cells of a healthy female buffalo (Mu-5579) that had
been established earlier in the laboratory were used.
The cryopreserved cells at passage 3–8 were thawed
and seeded in DMEM + 10% FBS in 25 mm2
tissue
culture flasks which were then incubated in a CO2 in-
cubator for 3–4 d till they reached 60–70% confluence
following which the cells were sub-cultured by
trypsinization. For preparation of non-viable cells, the
cells were suspended in Ca2+
- and Mg2+
-free DPBS
and were exposed to non-physiological temperatures,
i.e., 54, 56, 58, 60, or 62°C for 15, 30, 45, or 60 min
each in a water bath. The viability of cells was checked
by trypan blue staining for which the cell suspension
was incubated for 1–2 min with 0.4% trypan blue solu-
tion in phosphate-buffered saline, at room temperature.
The suspension was then immediately loaded on a he-
mocytometer and examined under a microscope at low
magnification. The percentage of dead cells was deter-
mined by the equation: (number of blue cells ÷ number
of total cells) × 100.
A piece of ear tissue was collected in a nearby abattoir from
buffaloes immediately after slaughter and was dipped in mus-
tard oil in a 50-ml Falcon tube. It was then transported to the
laboratory and kept at room temperature for 48 h. Before
isolation of cells, the ear tissue was wiped clean with blotting
paper after which the skin, along with the hair follicles, was
removed with a surgical blade. The tissue was washed thor-
oughly with sterile Ca2+
- and Mg2+
-free DPBS (DPBS-),
minced into small pieces (about 1 mm3
in size) using a sterile
surgical blade and then washed 4–5 times with DPBS contain-
ing 100 μg/ml gentamycin, 100 IU/ml penicillin and 100 μg/
ml streptomycin. The tissue pieces were then digested with an
enzyme cocktail containing 2 mg/ml collagenase, 1 mg/ml
hyaluronidase, and 0.05% trypsin in DMEM containing the
abovementioned antibiotics at 37°C in a shaking incubator
(185 rpm) for 3–4 h. The digested tissue was then filtered
through a nylon membrane filter (30 μm) and the resultant
cell suspension was centrifuged at 300×g for 5 min to obtain
a single cell suspension.
HMC, which included in vitro maturation, cumulus/zona
removal, manual enucleation, fusion, activation, and culture
of reconstructed embryos, was performed as described earlier
(Selokar et al. 2014). The blastocyst rate recorded on day 8 of
in vitro culture was taken as a measure of the developmental
competence of embryos. For examining the quality of blasto-
cysts, their total cell number (TCN) and level of apoptosis was
determined by TUNEL staining using In Situ Cell Death
Detection Kit, Fluorescein (11684795910 Roche, Germany)
as described earlier (Selokar et al. 2014). Cell counting was
performed from the digital images obtained on an inverted
Nikon fluorescence microscope. Each experiment was repeat-
ed at least 3 times. Apoptotic index (AI) = (number of TUNEL
DUAH ET AL.
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5. positive nuclei in the blastocyst/total number of nuclei count-
ed in that blastocyst) × 100.
Statistical analysis was carried out using Sigma Stat ver-
sion 3.1 (Aspire Software International, Ashburn, VA). The
datasets were analyzed by student’s t test. The percentage
values were analyzed after arcsine transformation. Data were
presented as mean ± SEM.
Results and discussion
We explored the possibility of producing embryos by HMC
from donor cells obtained from the tissue of freshly
slaughtered buffaloes. Because such cells could be a mixed
population of viable and non-viable cells, in the first part of
the study, we attempted producing HMC embryos from non-
viable cells since there was no such report earlier. We subject-
ed skin fibroblasts to various non-physiological temperatures
and found that exposure to 54°C was not so stressful, so cell
survival and dead rates were similar between 15 and 30 min,
although with different standard errors. Although exposure to
60°C was able to render all the cells non-viable, the minimum
duration of exposure required was 45 min. Since we wanted to
minimize the heat-induced damage to the cells, we selected
the lowest temperature and the shortest duration of exposure at
which all the cells were rendered non-viable. This was found
to be 62°C for 15 min (Fig. 1, Table 1). The non-viability of
the heat-treated cells was further confirmed by in vitro culture
(Loi et al. 2002). Briefly, following exposure of cells to 62°C
for 15 min, these were seeded in Falcon #25 flasks in
DMEM + 20% FBS following which, the attachment and
growth of cells was examined at 48 and 72 h. This was repeat-
ed for every trial. No cell attachment or growth was observed
in any trial. When we used these non-viable cells for HMC,
using the same lot of live cells as controls, the fusion and
cleavage rate was lower (P < 0.001) than that for the controls
(Table 2). Despite the donor cells being non-viable, the recon-
structed embryos developed to the blastocyst stage (Fig. 2c).
However, the blastocyst rate (P < 0.001) and TCN
(221.3 ± 25.14 vs 151.1 ± 21.69, P < 0.05) were lower and
the apoptotic index was higher (4.8 ± 1.06 vs 10.9 ± 1.21,
P < 0.001) for HMC embryos derived from non-viable cells
than that for the controls. The blastocyst rate could be low due
to heat treatment-induced damage to the donor cell plasma
membrane since reconstructed embryos derived from these
cells had a lower fusion and cleavage rate than that of controls.
This suggests that maintenance of plasma membrane integrity
to the extent that it is able to fuse with the demicytoplast
membrane is vital for forming a reconstructed embryo. The
poorer quality of blastocysts derived from non-viable cells, as
indicated by a lower TCN and higher apoptotic index than that
of controls, could be due to denaturation of or damage to some
thermolabile biomolecules, which may be involved in
reprogramming and subsequent embryonic development.
50µm
50µm
A BFig. 1 Skin fibroblast cells
subjected to trypan blue staining
(a) live cells before and (b) non-
viable cells after exposure to 62°C
for 15 min
Table 1 Percentage of live and dead cells, as indicated by trypan blue staining, following exposure to non-physiological temperatures
Temperature (°C) Duration of exposure (min)
15 30 45 60
Live (%) Dead (%) Live (%) Dead (%) Live (%) Dead (%) Live (%) Dead (%)
54 29.7 ± 6.06 70.3 ± 6.06 29.7 ± 2.33 70.3 ± 2.33 22.3 ± 4.66 77.7 ± 4.67 10.7 ± 3.52 86.0 ± 1.15
56 34.0 ± 6.66 66.0 ± 6.66 19.0 ± 3.60 81.0 ± 3.60 14.3 ± 0.67 85.7 ± 0.67 9.7 ± 1.85 90.3 ± 1.85
58 22.3 ± 5.78 77.7 ± 5.78 5.3 ± 1.45 94.7 ± 1.45 5.0 ± 0.58 95.0 ± 0.58 4.0 ± 0.0 96.0 ± 0.0
60 3.3 ± 0.88 96.7 ± 0.88 1.7 ± 0.33 98.3 ± 0.33 0.0 ± 0.0 100.0 ± 0.0 0.0 ± 0.0 100.0 ± 0.0
62 0.0 ± 0.0 100.0 ± 0.0 0.0 ± 0.0 100.0 ± 0.0 0.0 ± 0.0 100.0 ± 0.0 0.0 ± 0.0 100.0 ± 0.0
Data from 3 trials
Values are mean ± SEM
PRODUCTION OF HMC EMBRYOS FROM NON-VIABLE CELLS
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6. Major changes in the conformation of high-order DNA-pro-
tein complexes have been detected in the interphase and meta-
phase chromosomes between 55 and 75°C (Wolf et al. 1999).
SOX family of high-motility group proteins, which are key
regulators of embryonic development and are also involved in
the regulation of cellular differentiation, start to unfold at
greater than 46°C (Crane-Robinson et al. 1998). Finally, the
proteins of the nuclear matrix, which organize the DNA in
inactive or active operational domains, are the most thermally
labile proteins of the cells, undergoing denaturation at 43–
45°C (Roti Roti et al. 1998). Many of these families of pro-
teins could have undergone denaturation under the heat treat-
ment given by us and could have been degraded by the
proteolytic machinery of the embryo since unfolded, and de-
natured proteins are more readily degraded by them.
Many innovative approaches such as the use of cells cryo-
preserved by a simple method (Dong et al. 2004) or use of
donor cells after cooling to 4°C for 48 h (Hong et al. 2005)
have been used to widen the application of SCNT technology.
We developed a simple method based on storage of ear tissue
of animals in mustard oil at room temperature for 48 h for its
short-term preservation. This enabled harvesting somatic cells
for use as nuclear donors with very little damage to their
plasma membrane since they could be fused with the
demicytoplasts during HMC. Mustard oil was chosen based
on its good antibacterial quality (Turgis et al. 2009) and wide
Table 2 Developmental
competence of HMC embryos
produced from non-viable cells
Developmental stages Cell type
Live cells (control) Non-viable cells*
Reconstructs taken (n) 104 199
Reconstructs fused n (%) 97 (93.6 ± 1.72)a
131 (67.1 ± 2.83)b
Embryos cleaved n (%) 94 (90.3 ± 1.79)a
129 (65.8 ± 1.56)b
Reconstructs developed to n (%) 2- to 4-cell 29 (27.0 ± 2.98) 59 (30.4 ± 2.76)
8- to 16- cell 27 (27.0 ± 3.34) 54 (27.0 ± 2.05)
Morula 2 (2.1 ± 1.40) 3 (1.47 ± 0.75)
Blastocyst 36 (34.1 ± 2.43)a
13 (6.9 ± 2.18)b
Data from 9 trials
Values are mean ± SEM
Values with different superscripts within the same row differ significantly (P < 0.001)
500µm 500µm
50µm 20µm
A B
C D
Fig. 2 (a) Skin fibroblast cells
rendered non-viable by exposure
to 62°C for 15 min. (b) Cells ob-
tained from ear tissue of slaugh-
terhouse buffaloes and preserved
in mustard oil for 48 h at room
temperature. (c, d) Corresponding
blastocysts produced from these
cells
DUAH ET AL.
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7. availability with farmers in Asia, where buffaloes are reared.
Many, if not most of Asian farmers, do not have a refrigerator
to chill or freeze the tissue for short-term storage. The duration
of 48 h was chosen to enable transportation of tissue to the
laboratory. When the cells obtained after enzymatic digestion
of ear tissue were examined for viability by trypan blue ex-
clusion test and in vitro culture as explained above, these were
found to be non-viable. We could successfully produce
blastocyst-stage HMC embryos from cells harvested from
the ear tissue of slaughterhouse buffaloes following preserva-
tion of tissue in mustard oil. Although the blastocyst rate was
lower (P < 0.01) than that of controls, the blastocyst quality
was similar to that of controls as indicated by a similar TCN
(223.0 ± 27.89 vs 213.3 ± 28.21) and apoptotic index
(3.97 ± 0.67 vs 5.22 ± 0.51, Table 3, Fig. 2d). This is of
importance since the level of apoptosis of embryos is consid-
ered to be an important factor influencing the conception rate.
SCNT bovine embryos have been reported to exhibit higher
apoptotic morphology and/or TUNEL labeling compared to
their counterparts produced by in vitro fertilization (Selokar
et al. 2013).
Besides damage to the plasma membrane, the low blasto-
cyst rate in the above experiments could be due to DNA dam-
age in the treated cells and/or the cloned embryos produced.
DNA fragmentation has been shown to occur in 20% of re-
constructed embryos produced from freeze- dried cells, while
it was absent in control embryos (Loi et al. 2008). The ability
of donor cells, which had been rendered non-viable by heat
treatment or of cells preserved in mustard oil, to be
reprogrammed by demicytoplasts and develop to the
blastocyst stage suggests that the genomic integrity of many
of the cells was maintained even after these treatments. It is
also possible that some of the DNA damage that occurred due
to these treatments was repaired following fusion with the
demicytoplasts. Loi et al. (2008) reported that following the
use of freeze-dried sheep cells, 16% of the reconstructed em-
bryos developed to the blastocyst stage when 60% of donor
nuclei had obvious DNA damage, raising the possibility that
damaged DNA in the donor nucleus might be repaired by
factors present in the oocyte. In a recent study, DNA damage
was found to occur randomly in 40% of sheep lymphocytes,
whereas the genome was intact in the rest 60% of cells fol-
lowing lyophilization; however, lyophilized nuclei injected
into enucleated oocytes were repaired by a robust DNA
repairing activity of the oocytes and showed normal develop-
mental competence resulting in production of cloned embry-
os, which exhibited chromosome and cellular composition
comparable to those of embryos derived from fresh donor
cells (Iuso et al. 2013).
In conclusion, it was demonstrated in the present study that
HMC blastocysts can be produced from non-viable cells and
that maintenance of plasma membrane integrity of cells is vital
for forming reconstructed embryos.
Acknowledgments The present work was funded by the National
Agriculture Innovative Project (NAIP) grant to SKS (C 2-1-(5)/2007)
and MSC (C-2067 and 075). SKM and AS are recipients of CSIR-SRF
fellowship and TJS is a recipient of ICMR fellowship.
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