Development of caprine chimeric embryos reconstructed through ES-tetraploid complementation assay

Abstract

The aim of the present study was to evaluate the development of caprine chimeric embryos in different culture media, viz. RVCL and mCR2aa + 10% FBS. Inner cell mass (ICM) from hatched blastocysts of parthenogenetic activated embryos were used to produce ES cell-like cells (103) while 4-cell embryos obtained from IVF were utilized to produce tetraploid embryos (231). The aggregates prepared were randomly divided into 2 groups, viz. Gr 1 (RVCL) (53) and Gr 2 (mCR2aa +10% FBS) (50) followed by culture in humidified atmosphere of 5% CO2 at 38.5°C in a CO2 incubator. Outcome measures were aggregation, 8–16 cell, morula and blastocyst formation. The percentage of aggregation, 8–16 cell, morula and blastocyst in Gr 1 (RVCL) was 83.33±6.00%, 42.24±7.71%,29.59±7.64% and 11.49±5.36%, respectively while the percentage of aggregation, 8–16 cell, morula and blastocyst in Gr 2 (mCR2aa +10% FBS) was 91.66±4.32%, 54.76±7.75%, 22.32±5.75% and 14.58±5.18%, respectively. In conclusion, both the media supported the development of chimeric embryos up to blastocyst.

Full text

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Indian Journal of Animal Sciences 89(5): 519–521, May 2019/Short communication
Development of caprine chimeric embryos reconstructed through ES-tetraploid
complementation assay
JUHI PATHAK1, S D KHARCHE2 and ANJANA GOEL3
ICAR-Central Institute for Research on Goats, Makhdoom, Mathura, Uttar Pradesh 281 122 India
Key words: Caprine, Chimeric embryos, Culture media, ES-tetraploid complementation assay
Present address: 1PhD Scholar (juhi1690@gmail.com),
3Associate Professor (anjana.goel@gla.ac.in), Department of
Biotechnology, Institute of Applied Science and Humanities, GLA
University, Mathura, Uttar Pradesh. 2Principal Scientist (kharche1
@rediffmail.com), Animal Physiology and Reproduction
Division.
The first mouse embryonic chimeras were generated
independently by Tarkowski through aggregating 2 eight-
cell embryos (Tarkowski 1961). Since then, chimerism has
been experimentally induced in many species of mammals
including sheep (Butler et al. 1987), goats (Jia et al. 2008),
bovine (Razza et al. 2016), pigs (Nagashima et al. 2004)
and cattle (Saito et al. 2003).
Attempts to improve chimeric embryo production by ES-
tetraploid complementation have met with little success.
As RVCL and mCR2aa are widely used for embryo culture
media for parthenogenetically activated and IVF derived
embryos (Kharche et al. 2011, Kharche et al. 2016, Pathak
et al. 2017), this prompted us to evaluate the developmental
potency of chimeric embryos in RVCL and mCR2aa +10%
FBS. To the best of our knowledge, the effects of
readymade/complex culture medium and chemically
defined medium on developmental potency of chimeric goat
embryos have not been reported.
Collection of ovaries, recovery of oocytes and in vitro
maturation (IVM) was done as per the method of Kharche
et al. (2016). In vitro fertilization was carried out as per the
method described by Kharche et al. (2011) with slight
modifications. For tetraploid embryo production, fertilized
two cell embryos (609) were equilibrated in fusion buffer
(0.3 M mannitol solution containing 0.1 mM MgSO4.7H2O,
0.05 mM CaCl2. 2H2O, 0.5 mM HEPES and 1 mg/ml BSA)
for 5–10 sec before they were placed into a 0.5 mm gap
BTX micro slide fusion chamber (BTX, ECM 2001) filled
with fusion buffer in such a manner that the inter-
blastomeric axis was positioned in parallel to the electrodes.
Two-cell embryos were aligned between electrodes using
an AC field of 5V and 5s, and fusion of blastomeres was
induced at 38.5°C by a single DC pulse for 1.2 kV/cm for
4µs with 3s post fusion AC. After the electric pulse, the
fused embryos were scored and cultured in RVCL media
supplemented with 1% BSA for 48 h in humidified
atmosphere of 5% CO2 at 38.5ºC in CO2 incubator.
Collection of ovaries, recovery of oocytes and in vitro
maturation (IVM) was done as per method described for
the production of tetraploid fertilized embryos. After 27 h
of in vitro maturation, denuded oocytes (n=2628) were then
chemically activated with 5 µM calcium ionophore for 5 to
7 min followed by 4 horticulture in embryo development
medium (RVCL) containing 2 mM DMAP. After 4 h of
culture, the oocytes were washed and cultured in RVCL.
These embryos were developed up to expanded to hatched
blastocyst stage in embryo development medium in
humidified atmosphere at 38.5°C with 5% CO2 in CO2
incubator.
For stem cell production, inner cell mass (ICM) was
mechanically isolated from hatched blastocysts and were
cultured on Mitomycin-C inactivated goat fetal fibroblasts
feeder layer in stem cell culture medium. Subsequent
colonies were passaged every 4–5 day and media was
replaced after every 24 h.
A pair of zona-free tetraploid embryos and one clump
of pESCs at passage 2 and 3 were randomly distributed for
aggregation in different culture media. In Gr 1 the
aggregates were cultured on granulosa cumulus cells
monolayer (GC) in RVCL in humidified atmosphere at
38.5°C with 5% CO2 in CO2 incubator while in Gr 2
aggregates were cultured on granulosa cumulus cells
monolayer (GC) in mCR2aa medium (10% FBS) in
humidified atmosphere at 38.5°C with 5% CO2 in CO2
incubator.
A total of 609 cleaved two-cell caprine IVF embryos
were subjected to electrofusion at 1.2 kV/cm for 4µs to
produce tetraploid embryos. The percentage of fusion was
85.26±1.55% and fused embryos were further developed
upto 4-cell stage as 4-cell embryos formed were utilized
for chimeric embryo production. Cleavage rate was
57.89±2.60% and the percentage of 2-cell and 4-cell embryo
were 23.27±3.28 and 71.31±3.78, respectively.
The embryo development among RVCL (Gr 1) and
mCR2aa medium (Gr 2) was compared. In Gr 1, the
percentage of aggregation efficiency, aggregates at 8–16
cell, morula and blastocyst were 83.33±6.00%,
42.24±7.71%, 29.59±7.64% and 11.49±5.36%, respectively
while in Gr 2, the percentage of aggregation efficiency,
aggregates at 8–16 cell, morula and blastocyst (Fig. 1A-F)
were 91.66±4.32%, 54.76±7.75, 22.32±5.75 and
14.58±5.18% (Table 1).
https://doi.org/10.56093/ijans.v89i5.90018

520 PATHAK ET AL. [Indian Journal of Animal Sciences 89 (5)
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Table 1. Es-tetraploid complementation assay in different culture media
Group No. of tetraploid No. of Aggregation Aggregates at 8–16 Morula Blastocyst
embryos aggregates efficiency (%) cell stage (%) (%) (%)
RVCL 113 53 43a (83.33±6.00%) 21a (42.24±7.71%) 16a (29.59±7.64%) 6a (11.49±5.36%)
mCR2 aa 118 50 46a (91.66±4.32%) 27a (54.76±7.75%) 12a (22.32±5.75%) 7a (14.58±5.18%)
Values within different superscripts in the same column are significantly different (P<0.05).
Fig. 1(A-F). Generation of chimeric embryos. A. In vitro
fertilized 2-cell embryos (2n), B. Tetraploid morula (4n), C.
Parthenogenetic hatched blastocyst (2n), D. Parthenogenetic
pESCs (2n), E. Aggregate of pESCs (2n) and tetraploid morula
(4n), F. Chimeric blastocyst.
Fig. 2. Tetraploid chromosome.
The composition of the embryo culture medium can
directly influence embryo developmental capacity and
blastocyst gene expression. Readymade/complex culture
medium in vitro embryo production systems are believed to
provide necessary growth factors and (or) metabolize
embryo toxic factors inhibitory to development while for
defined medium, stimulatory effects of serum
supplementation (Gutierrez-Adan et al. 2001), essential and
non-essential amino acids on rates of embryonic
development are well described. The supplementation of
basal culture media with animal serum of different origins
is essential for cell growth, metabolism, and to stimulate
proliferation. Fetal bovine serum (FBS) is a cocktail of most
of the factors required for cell attachment, growth and
proliferation and is thus used as an almost universal growth
supplement effective for most types of human and animal
cells.
Our results indicate that the aggregate efficiency, 8–16
cell stage and blastocyst formed in mCR2aa + 10% FBS
were comparatively higher than in RVCL media while
morula formation in mCR2aa +10% FBS was comparatively
lower than in RVCL media. Higher number of morula in
RVCL media might be due to the fact that more number of
embryos got arrested at morula stage and did not develop
to blastocyst while in mCR2aa +10% FBS more number of
embryos cleared embryonic arrest at morula stage and
developed to blastocysts. It can be concluded that both the
media support the development of chimeric embryos up to
blastocyst stage.
SUMMARY
The aim of the present study was to evaluate the
development of caprine chimeric embryos in different
culture media, viz. RVCL and mCR2aa + 10% FBS. Inner
cell mass (ICM) from hatched blastocysts of
parthenogenetic activated embryos were used to produce
ES cell-like cells (103) while 4-cell embryos obtained from
IVF were utilized to produce tetraploid embryos (231). The
aggregates prepared were randomly divided into 2 groups,
viz. Gr 1 (RVCL) (53) and Gr 2 (mCR2aa +10% FBS) (50)
followed by culture in humidified atmosphere of 5% CO2
at 38.5°C in a CO2 incubator. Outcome measures were
aggregation, 8–16 cell, morula and blastocyst formation.
The percentage of aggregation, 8–16 cell, morula and
blastocyst in Gr 1 (RVCL) was 83.33±6.00%, 42.24±7.71%,

May 2019] GENERATION OF CAPRINE CHIMERIC EMBRYOS 521
45
29.59±7.64% and 11.49±5.36%, respectively while the
percentage of aggregation, 8–16 cell, morula and blastocyst
in Gr 2 (mCR2aa +10% FBS) was 91.66±4.32%,
54.76±7.75%, 22.32±5.75% and 14.58±5.18%, respectively.
In conclusion, both the media supported the development
of chimeric embryos up to blastocyst.
ACKNOWLEDGEMENTS
The authors wish to thank Director, ICAR-C I R G,
Makhdoom for providing the facilities needed. This study
was financially supported by ICAR-NASF
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