Vol.1, No.3, 97-105 (2011)
doi:10.4236/ojas.2011.13013
C
opyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/OJAS/
Open Journal of Animal Sciences
Development and quality of bovine embryos produced
in vitro using growth factor supplemented serum-free
system
Arindam Dhali, Vahida M. Anchamparuthy, Steve P. Butler, Isis K. Mullarky, Ronald E. Pear-
son, Frank C. Gwazdauskas*
Department of Dairy Science, Virginia Polytechnic Institute and State University, Blacksburg, USA;
*Corresponding Author: guaz@vt.edu
Received 1 July 2011; revised 10 August 2011; accepted 31 August 2011.
ABSTRACT
The influence of a growth factor supplemented
serum-free system on the development, gene
expression, and cryotolerance of in vitro pro-
duced bovine embryos was investigated. To
assess the embryo development and gene ex-
pression in blastocysts, abattoir-derived oo-
cytes (obtained from 3 - 10 or <3 mm follicles)
were matured and fertilized in serum-free media
and cultured in synthetic oviductal fluid sup-
plemented with fetal bovine serum (FBS, 4%),
epidermal growth factor (EGF, 10 ng/mL), insulin
like growth factor-1 (IGF-1, 100 ng/mL), stem cell
factor (SCF, 50 ng/mL) or combinations of the
growth factors. Expressions of selected gene
transcripts were relatively quantified in the d 8
blastocysts. To assess the cryotolerance, d 4
morulae (derived from 3 - 10 mm follicles and
cultured with the supplementation of FBS or
combinations of the growth factors) were vitri-
fied, thawed and cultured (with respective sup-
plementations). Total cell number and DNA frag-
mentation in blastocysts derived from the vitri-
fied morulae were assessed through TUNEL
assay. The rate (%) of cleavage, blastocyst and
expanded/hatched blastocyst did not differ
among the culture medium supplementations
within the follicle size of 3 - 10 mm (range 65.1 ±
4.3 - 75.4 ± 3.9; 22.4 ± 3.9 - 36.4 ± 3.6; and 11.2 ±
2.9 - 23.3 ± 3.2, respectively) or <3 mm (range
59.3 ± 4.2 - 74.5 ± 3.7; 15.0 ± 3.5 - 28.7 ± 4.5; and
9.3 ± 2.8 - 17. 3 ± 2. 7, re sp ecti vely). Nevert heles s,
significantly lower (P < 0.05) cleavage and
blastocyst rates with FBS and lower blastocyst
rate with SCF supplementations were observed
for the oocytes derived from <3 compared to 3 -
10 mm follicles. The expression patterns of
BCL-2, BAX, HSP1A1, GJA1 and BIRC5 tran-
script s varied significantly (P < 0.05) in all cases,
except for BIRC5 in the blastocysts derived
from 3 - 10 mm follicles. Following thaw and
culture, the dev elopme nt (% ) of vi trified morulae
into expanded/hat ch ed bl a sto cyst s was lower (P
< 0.01) with the supplementation of growth fac-
tors compared to FBS. In contrast, total number
of cells and DNA fragmentation index in blas-
tocysts were not different among the treatments.
In conclusion, the growth factor supplemented
serum-free system was satisfactory for in vitro
bovine embryo production. Nevertheless, the
system was not efficient when embryos were
derived from <3 mm follicles and cultured with
SCF. Additionally, gene expression patterns and
cryotoler ance of the emb ry os were affec ted with
the treatments of growth factors compared to
serum.
Keywords: IVF; Growth Factors; Embryo
Development; Gene Expression; Cryotolerance;
Bovine
1. INTRODUCTION
Although the intrinsic quality of oocytes determines
embryo quality, the microenvironment of the in vitro
production system largely influences the development
and quality of embryos [1-5]. A wide range of culture
systems supports the in vitro development of preimplan-
tation embryos [6]. Serum supplemented systems have
been used widely for in vitro embryo production. Nev-
ertheless, the composition of serum and its effects on
embryo development are variable. A prolonged exposure
to serum may alter embryo quality and affect fetal de-
velopment [5]. A chemically defined serum-free system
is advantageous for in vitro embryo production as exclu-
A. Dhali et al. / Open Journal of Animal Sciences 1 (2011) 97-105
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98
sion of serum in the media avoids its negative influences
on embryo development and quality [7]. Hence, the em-
bryos produced in a serum-free system can serve as
valuable model for many research applications.
Previous attempts have assessed the beneficial effects
of growth factors on the development and quality of in
vitro produced embryos. Insulin like growth factor-1
(IGF-1) improves the survival of embryos [8] and serves
as an anti-apoptotic factor during bovine oocyte matura-
tion and embryo development [9,10]. Moreover, IGF-1
protects preimplantation embryos against heat shock
[11]. Epidermal growth factor (EGF) exerts a positive
effect on blastocyst cell number, but it may not serve as
an anti-apoptotic factor [9]. Stem cell factor (SCF) en-
hances the development of embryos into late blastocyst
and hatching stages [12] and improves the quality of
embryos cultured with 500 ng/mL Fas-L [13]. It is evi-
dent that IGF-1, EGF and SCF protect embryos from
oxidative stress [14].
In vitro produced embryos are exposed to sub-optimal
conditions that greatly differ from the in vivo environ-
ment. The composition of culture medium mimicking
the in vivo environment including growth factors is cru-
cial to enhance embryo development in vitro [15]. The
development and quality of synthetic oviductal fluid
(SOF) generated embryos are more similar to those of in
vivo embryos than are those embryos produced in tissue
culture medium 199 (TCM 199) [2]. The efficiency of an
in vitro embryo production system is traditionally as-
sessed on the basis of cleavage of fertilized oocytes and
their further development into blastocysts. Nevertheless,
a simultaneous assessment of embryo quality would be
advantageous for understanding the influence of the mi-
croenvironment of such a system. Cryotolerance and the
expression pattern of development and apoptosis related
genes are useful indicators of the quality of embryos
produced in vitro [5,6].
Currently, insufficient information is available on the
effect of growth factor supplemented serum-free systems
on the development and quality of in vitro produced bo-
vine embryos. In this study, we assessed the influence of
serum-free IVM and IVF, and growth factor (IGF-1,
EGF, SCF) supplemented serum-free IVC on the devel-
opment, gene expression, and cryotolerance of bovine
embryos.
2. MATERIALS AND METHODS
The chemicals used in the experiments were procured
from Mallinckrodt Baker Inc., Phillipsburg, NJ, USA
unless mentioned otherwise. The growth factors (IGF-1,
EGF, SCF), essential amino acids solution, non essential
amino acids solution, TCM 199 and gentamicin sulfate
solution were procured from Invitrogen Corporation,
Carslbad, CA, USA. Fetal bovine serum (FBS), bovine
serum albumin (BSA, fraction V, fatty acid free), FSH,
LH, 17β-estradiol, sodium pyruvate, L-glutamine, heap-
rin, and phenol red solution were procured from Sigma-
Aldrich, St. Louis, MO, USA. The same BSA brand was
used for performing IVM, IVF and IVC during the
study.
2.1. Design of Experiment
2.1.1. Experiment-1
In this experiment, the effect of growth factor sup-
plemented serum-free system on the development and
gene expression of in vitro produced bovine embryos
was assessed. Oocytes were aspirated from 3 - 10 or <3
mm follicles and matured and fertilized in serum-free
media. Presumptive zygotes were cultured in growth
factor supplemented serum-free medium. The develop-
ment of embryos was recorded during the culture period
and expressions of the development and apoptosis re-
lated gene transcripts were relatively quantified in the d
8 blastocysts. The oocytes were grouped on the basis of
their follicle size as low developmental competence is
evident for the bovine oocytes derived from <3 mm fol-
licles [16,17].
2.1.2. Experiment-2
In this experiment, cryotolerance and apoptosis of the
morulae produced in vitro using the growth factor sup-
plemented serum-free system were assessed. Oocytes
were aspirated from 3 - 10 mm follicles, matured, fertile-
ized, and cultured as mentioned above. Morulae were
vitrified, thawed, and cultured and the post-thaw devel-
opment and apoptosis in the embryos were assessed.
2.2. In Vitro Embryo Production
Previously we have reported the detailed procedures
of in vitro bovine embryo production using a serum-free
SOF based system [18]. In brief, oocytes were aspirated
from 3-10 or <3 mm follicles of abattoir-derived ovaries.
IVM of the oocytes was conducted for 22 to 24 h in
TCM 199 supplemented with BSA (8 mg/mL), LH (0.02
U/mL), FSH (0.02 U/mL) and 17β-estradiol (1 μg/mL).
Sperm samples (provided by two bulls) were prepared
for IVF through the swim-up separation method in heap-
rin (10 µg/mL) and BSA (8 mg/mL) supplemented
HEPES buffered SOF-IVF medium. Matured oocytes
were washed and placed into 48 µL drops (10 oo-
cytes/drop) of heparin and BSA supplemented SOF-IVF
under mineral oil. A prepared sperm sample (2 µL) was
added into each drop for a final volume of 50 µL and
concentration of 1 × 106 sperm/mL. Cumulus cells were
removed from the oocytes after an 18 h of sperm-oocyte
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99
incubation and presumptive zygotes were cultured in the
drops of SOF-IVC medium supplemented with BSA (8
mg/mL, in all experimental groups) and FBS (4%),
IGF-1 (100 ng/mL), EGF (10 ng/mL), SCF (50 ng/mL),
EGF (10 ng/mL) + IGF-1 (100 ng/mL), EGF (10 ng/mL)
+ SCF (50 ng/mL) or IGF-1 (100 ng/mL) + SCF (50
ng/mL). During IVM, IVF and IVC, the oocytes or em-
bryos were incubated in a humidified 5% CO2 atmos-
phere at 38.5˚C. The development of embryos was re-
corded on d 4 (~114 h post-insemination) and d 8 (~210
h post-insemination) of IVC.
2.3. Quantitative Real-Time PCR Analysis
The expression of development and apoptosis related
gene transcripts in blastocysts was relatively quantified
by reverse transcription and real-time quantitative PCR
methods. In brief, d 8 blastocysts (~210 h post-insemi-
nation) were collected, washed in Dulbeccos PBS (Invi-
trogen Corporation) supplemented with 0.1% polyvinyl
alcohol and stored in pools at –80˚C until processed for
RNA extraction. Total RNA was extracted from blasto-
cysts using Absolutely RNA® Microprep Kit (Stratagene,
La Jolla, CA, USA). Purified RNA was transcribed into
cDNA immediately using High-Capacity cDNA Archive
Kit (Applied Biosystems, Foster City, CA, USA) and
stored at –20˚C until used for PCR analysis.
Real-time PCR was performed in a 7300 Real-Time
PCR System (Applied Biosystems) using SYBR® Green
detection protocol. Briefly, cDNA (equivalent to 4 ng of
total RNA), 0.2 µM of each primer and 1 × SYBR®
Green Mix (Applied Biosystems) were used in a total
volume of 25 µL. When the reaction was completed,
melting curve analysis was performed to confirm the
product purity. Specific primers were designed and used
to amplify ACTB (β-actin) (sense 5’-CTCTTCCAG
CCTTCCTTCCT-3’ and antisense 5’-GGGCAGTGA
Tctctttctgc-3’, 178 bp amplicon), BAX (sense
5’-TCTGACGGCAACTTCAACTG-3’ and antisense
5’-TGGGTGTCCCAAAGTAGGAG-3’, 205 bp ampli-
con), BCL-2 (sense 5’-ATGACTTCTCTCGGCGCTAC-
3’ and antisense 5’-CGGTTCAGGTACTCGGTCAT-3’,
244 bp amplicon), HSPA1A (heat shock protein 70) (sense
5’-CAAGATCACCATCACCAACG-3’ and antisense
5’-AAATCACCTCCTGGCACTTG-3’, 239 bp amplicon),
GJA1 (connexin 43) (sense 5’-GGACATGCACTTGA-
AGCAGA-3’ and antisense 5’-TGTAAACGGCACTCA-
AGCTG-3’, 188 bp amplicon) and BI RC5 (survivin)
(sense 5’-CCTGGCAGCTCTACCTCAAG-3’ and an-
tisense 5’-GAAAGCACAACCGGATGAAT-3’, 233 bp
amplicon), The relative abundance of gene transcripts
was calculated using the 2-ΔΔCt method and β-actin as
endogenous reference gene [6,19].
2.4. Vitrification, Thawing and Culturing of
Embryos
Morulae were produced in vitro from the oocytes as-
pirated from 3 - 10 mm follicles. Oocytes were matured,
fertilized, and presumptive zygotes were cultured for 4 d
in the drops of SOF-IVC medium supplemented with
BSA (8 mg/mL, in all experimental groups) and FBS
(4%), EGF (10 ng/mL)+IGF-1 (100 ng/mL), EGF (10
ng/mL) + SCF (50 ng/mL) or IGF-1 (100 ng/mL)+SCF
(50 ng/mL). The d 4 (~114 h post-insemination) morulae
were vitrified using the modified droplet vitrification
method described previously [19]. Composition of the
vitrification solution (VS) was 17.5% ethylene glycol,
17.5% dimethyl sulfoxide, 0.5 M sucrose and 4 mg/mL
BSA in TCM-199 medium. Briefly, 6 to 8 embryos were
equilibrated in the equilibration medium (50% VS in
TCM-199) for 3 min and subsequently washed in VS
three times. The embryos along with VS were aspirated
into a pipette. The pipette was held horizontally and a
drop (~5 µL) containing embryos in VS was formed at
the tip. The pipette was then held at 45 above the liq-
uid nitrogen level (~5 cm) and the drop was placed onto
liquid nitrogen by applying a gentle tweak and liquid
nitrogen was poured over the drop. The drop was vitri-
fied and sank into liquid nitrogen immediately. The in-
terval between the contact of embryos with VS and cool-
ing did not exceed 45 s. Thawing and removal of cryo-
protectants were done by placing the vitrified drop di-
rectly into dilution medium (0.3 M sucrose and 4 mg/mL
BSA in TCM-199 medium) for 3 min and then transfer
into SOF-IVC medium for 5 min. Morphologically nor-
mal embryos were subsequently washed in SOF-IVC
medium and cultured for 4 d in the drops of SOF-IVC
supplemented with BSA and FBS or respective growth
factors as stated previously. A morula was considered to
have survived following vitrification and thawing when
it cleaved further during the post-thaw culturing (~24 h
post-thaw). Development of vitrified morulae into blas-
tocysts or expanded/hatched blastocysts was recorded at
the end of culturing (~96 h post-thaw).
2.5. Detection of DNA Fragmentation by
TUNEL Assay
The TUNEL assay was performed using the In Situ
Cell Death Detection Kit (Fluorescein; Roche Diagnos-
tics Corp., Indianapolis, IN, USA) according to the
method described previously [20] after suitable modify-
cations. Embryos were stained on microscopic slides and
washed in SOFH-IVF during the procedure. Briefly,
blastocysts derived from the vitrified morulae were
washed four times and fixed in paraformaldehyde solu-
tion (4% w/v in PBS, pH 7.4) for 1 h at room tempera-
A. Dhali et al. / Open Journal of Animal Sciences 1 (2011) 97-105
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100
ture. Embryos were washed and incubated in permeabi-
lization solution [0.5% (v/v) Triton X-100, 0.1% (w/v)
sodium citrate] for 30 min at room temperature. After
washing, positive and negative control embryos were
incubated in DNase (50 U/mL) at 37˚C for 20 min. Em-
bryos were washed and the treatment and positive con-
trol embryos were incubated in TUNEL reaction mixture
in the dark for 1 h at 37C in a humidified chamber. The
negative control embryos were incubated in the absence
of the enzyme terminal transferase. Following the TUNEL
reaction, the embryos were washed and incubated in
RNase A (50 µg/mL) for 1 h at room temperature in the
dark. Embryos were incubated in propidium iodide (0.5
µg/mL; Roche Diagnostics Corp.) for 30 min at room
temperature in the dark to label all nuclei. The embryos
were washed to remove excess propidium iodide and a
cover slip was mounted over the samples. The stained
embryos were examined under a Zeiss LSM 510 laser
scanning microscope. Positive reactivity was indicated
by a bright green/yellow fluorescence indicating cells
with DNA fragmentation and the total cell number was
determined by red fluorescence. The DNA fragmentation
index was calculated by dividing the number of TUNEL-
positive cells by the total number of cells per embryo.
2.6. Statistical Analysis
Data were analyzed using the SPSS 14.0 software
package (SPSS Inc., Chicago, Illinois, USA) and pre-
sented as mean ± SEM. In the Experiment-1, variations
in the occurrence of different development stages were
analyzed among treatments (IVC medium supplementa-
tion) for each follicle size (3 - 10 or <3 mm) separately.
Each embryo was assigned a score of either 1 (embryo
developed into the stage of interest) or 0 (embryo did not
develop into the stage of interest) and subjected to
ANOVA followed by multiple pairwise mean compare-
sons using Student-Newman-Keuls (SNK) test [18]. In
addition, variations in the occurrence of different devel-
opment stages were analyzed between the follicle sizes
for each IVC medium supplementation separately. Each
embryo was given a score as stated above and subjected
to ANOVA. Variations in the expression of different
gene transcripts were analyzed by Student’s t-test. In the
Experiment-2, variations in the post-thaw development
of vitrified embryos were analyzed among treatments
(IVC medium supplementation). Each embryo was given
a score as stated above and subjected to ANOVA fol-
lowed by multiple pairwise mean comparisons using
SNK test. Variations in the DNA fragmentation index
and total number of cells in the TUNEL-stained embryos
were analyzed among treatments (IVC medium supple-
mentation) by ANOVA followed by multiple pairwise
mean comparisons using SNK test.
3. RESULTS
3.1. In Vitro Embryo Development
The experiment was performed in three replicates to
assess the effect of growth factor supplemented serum-
free system on embryo development (Table 1). The rates
of cleavage, morula, blastocyst and expanded/ hatched
blastocyst development did not differ among the IVC
medium supplementations within a follicle size (3 - 10
or <3 mm). Nevertheless, comparisons of embryo de-
velopment between the follicle sizes for each IVC me-
dium supplementation revealed lower (P < 0.05) cleav-
age and blastocyst rates with FBS and blastocyst rate
with SCF supplementations for the oocytes derived from
<3 compared to 3 - 10 follicles.
3.2. Expression of Development and
Apoptosis Related Gene Transcripts
Three replicate PCR experiments were conducted to
relatively quantify the expression of all genes. Total
RNA was purified from 15 to 20 blastocysts from the
three experimental pools for each treatment. The expres-
sion of different gene transcripts in the d 8 blastocysts
cultured with growth factor supplementations is depicted
relative to that of the embryos cultured with FBS (Fig-
ure 1). BCL-2 expression was increased (P < 0.05) with
EGF, IGF-1 and EGF + IGF-1 treatments for 3 - 10 mm
follicles and IGF-1, EGF + SCF and IGF-1 + SCF treat-
ments for <3 mm follicles. BAX expression was in-
creased (P < 0.05) when embryos were treated with
growth factors irrespective of follicle size except for <3
mm follicles and EGF + IGF-1 treatment. Greater (P <
0.05) HSPA1A expression was observed in the embryos
derived from 3 - 10 mm follicles with EGF + IGF-1 sup-
plementation or <3 mm follicles with EGF supplementa-
tion. The expression of GJA1 was (P < 0.05) low when
embryos were treated with growth factors irrespective of
follicle size except for 3 - 10 mm follicles and IGF-1
treatment. Although similar BIRC5 expression was ob-
served in the embryos derived from 3 - 10 mm follicles
with growth factor treatments, it was less (P < 0.05) in
the embryos derived from <3 mm follicles with EGF,
EGF + SCF and IGF-1 + SCF treatments.
3.3. Development and Apoptosis of
Embryos Following Vitrification,
Thawing and Culturing
The experiment was performed in six replicates to as-
sess the development of vitrified morulae in different
treatment groups (Ta b l e 2 ). Embryo development in all
the vitrified groups was less (P < 0.01) compared with
that of non vitrified controls. Among the vitrified groups,
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101
Ta ble 1 . In vitro development (mean ± SEM) of bovine embryos. Oocytes were aspirated from 3 - 10 or <3 mm follicles, matured
and fertilized in serum-free media and embryos were cultured in serum (FBS, 4%) or growth factor (EGF, 10 ng/mL; IGF-1, 100
ng/mL; SCF, 50 ng/mL) supplemented medium.
Experimental group Oocytes fertilized (n) Cleavage (% )Morulae (%)Blastocysts (%) Expanded/hatched blastocysts (%)
3-10 mm follicles
FBS 176 73.9 ± 3.3* 51.1 ± 3.8 36.4 ± 3.6§ 23.3 ± 3.2
EGF 116 71.6 ± 4.2 28.5 ± 4.2 22.4 ± 3.9 11.2 ± 2.9
IGF-1 137 74.5 ± 3.7 48.2 ± 4.3 28.5 ± 3.9 16.1 ± 3.1
SCF 111 69.4 ± 4.4 33.3 ± 4.5 26.1 ± 4.2$ 14.4 ± 3.3
EGF+IGF-1 139 74.8 ± 3.7 46.0 ± 4.2 34.5 ± 4.0 20.9 ± 3.5
EGF+SCF 122 75.4 ± 3.9 42.6 ± 4.5 24.6 ± 3.9 15.6 ± 3.3
IGF-1+SCF 126 65.1 ± 4.3 44.4 ± 4.4 27.8 ± 4.0 16.7 ± 3.3
<3 mm follicles
FBS 202 64.4 ± 3.4* 41.1 ± 3.5 26.2 ± 3.1§ 17.3 ± 2.7
EGF 101 68.3 ± 4.7 28.7 ± 4.5 28.7 ± 4.5 16.8 ± 3.7
IGF-1 123 71.5 ± 4.1 40.7 ± 4.4 22.8 ± 3.8 16.3 ± 3.3
SCF 107 59.8 ± 4.8 23.4 ± 4.1 15.0 ± 3.5$ 9.3 ± 2.8
EGF+IGF-1 130 70.8 ± 4.0 39.2 ± 4.3 25.4 ± 3.8 14.6 ± 3.1
EGF+SCF 137 74.5 ± 3.7 32.1 ± 4.0 27.7 ± 3.8 16.8 ± 3.2
IGF-1+SCF 140 59.3 ± 4.2 35.0 ± 4.0 19.3 ± 3.3 12.1 ± 2.8
*,§,$ indicates that for the same symbol values differ significantly (P < 0.05) between the follicle sizes for the given culture medium supplementation; Effect of
treatments was not significant within the groups (3 - 10 or <3 mm follicles).
Figure 1. Variations (mean ± SEM) in the relative expression of BAX, BCL2, HSPA1A (Hsp70), GJA1 (connexin - Cx43) and BIRC5
(survivin) transcripts in the d 8 bovine blastocysts produced in vitro. Oocytes were aspirated from 3 - 10 or <3 mm follicles, matured
and fertilized in serum-free media and embryos were cultured in serum (FBS, 4%) or growth factor (EGF, 10 ng/mL; IGF-1, 100
ng/mL; SCF, 50 ng/mL) supplemented medium. a-d on error bar indicates a significant difference (P < 0.05).
Openly accessible at
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102
Table 2. Cryotolerance of bovine morulae produced in vitro. Oocytes were aspirated from 3-10 mm follicles, matured and fertilized
in serum-free media and embryos were cultured in serum (FBS, 4%) or growth factor (EGF, 10 ng/mL; IGF-1, 100 ng/mL; SCF, 50
ng/mL) supplemented medium. Day-4 morulae were vitrified and post-thaw culturing was done for 4 d.
Treatment Morulae
vitrified (n)
Morulae
recovered after
thawing (n)
Damaged
Morulae after
thawing (n)
Morulae
cultured
(n)
Morulae
survived
(% ± SEM)
Blastocysts
(% ± SEM)
Expanded/hatched
blastocysts
(% ± SEM)
Not vitrified control
FBS - - - 96 90.6 ± 3.0A60.4 ± 5.0A 44.8 ± 5.1A
Vitrified
FBS 109 106 3 103 75.7 ± 4.2B44.7 ± 4.9B 28.2 ± 4.5B
EGF + IGF-1 110 109 7 102 67.0 ± 4.7BC 33.0 ± 4.7BC 15.5 ± 3.6C
EGF + SCF 110 108 6 102 57.3 ± 4.9C29.1 ± 4.5C 12.6 ± 3.3C
IGF-1 + SCF 111 109 6 103 61.2 ± 4.8BC 22.3 ± 4.1C 9.7 ± 2.9C
A,B,C indicates values within column differ at P < 0.01.
following thaw and culture, lower (P < 0.01) embryo
survival with EGF+SCF and blastocyst rate with EGF +
SCF and IGF-1+SCF supplementations were observed
compared to FBS. The development rate of ex-
panded/hatched blastocyst was lower (P < 0.01) with
growth factor supplementations compared to FBS. To
determine the total number of cells and DNA fragmenta-
tion in blastocysts, five embryos in each treatment group
were subjected to TUNEL assay. Total number of cells
was greater (P < 0.05) in the non vitrified control group,
but it was similar among the vitrified groups (Figure 2).
Similarly, the DNA fragmentation index was lowest (P <
0.05) in the non vitrified control group, but it was not
different among the vitrified groups (Figure 2).
4. DISCUSSION
The results indicated that the described growth factor
supplemented serum-free system was satisfactory for in
vitro bovine embryo production. Nevertheless, the sys-
tem was not efficient when embryos were derived from
<3 mm follicles and cultured with SCF supplementation.
Additionally, gene expression and cryotolerance of the
embryos were affected with the treatments of growth
factors compared to serum.
In the present study, oocytes were obtained from 3 -
10 or <3 mm follicles, matured and fertilized in serum-
free media and subsequently cultured in serum or growth
factor supplemented medium. It is generally concluded
that oocytes from larger follicles have a greater devel-
opment competence than oocytes from smaller follicles
[21]. Rate of normal fertilization is lower for the oocytes
derived from 1 - 2 compared to 2 - 4 or 4 - 8 mm folli-
cles [22]. Blastocyst yield is lower for the oocytes de-
rived from 2 - 6 than >6 mm follicles [23]. Oocytes from
3 mm follicles either do not develop past the 16-cell
stage [16] or yield fewer blastocysts compared to oo-
cytes from >3 mm follicles [17]. In our study, within a
follicle size (3 - 10 or <3 mm), similar embryo devel-
opment was ob- served irrespective of culture medium
supplementation. The results indicated that the described
growth factor supplemented serum-free system was sat-
isfactory for in vitro bovine embryo production using 3 -
10 mm follicles. The comparisons of embryo develop-
ment between the follicle sizes within a culture medium
supplementation revealed similar embryo development
irrespective of follicle size for all the supplementations
except FBS and SCF. The development was significantly
lower with FBS (cleavage and blastocyst) and SCF
(blastocyst) supplementations when embryos were de-
rived from <3 compared to 3 - 10 mm follicles. The re-
sults indicated that FBS and SCF supplementations were
not efficient for embryo production from the oocytes
derived from <3 mm follicles, while similar blastocyst
and expanded/hatched blastocyst rates irrespective of the
source of oocytes were seen in our previous study [18].
The expression pattern of development and/or apop-
tosis related genes reflects embryo quality [2,24,25].
BIRC5 (survivin) is important for optimal embryo de-
velopment and it suppresses apoptosis in preimplantation
embryos [25]. Although HSPA1A is a constitutive gene
in embryos, its expression level is linked to the response
to cellular stress [26]. It is believed that the relative ratio
between proapoptotic (BAX) and antiapoptotic (BCL-2)
proteins determines the fate of blastomeres [27], but
these proteins also function independently to regulate
cell death [28]. GJA1 (connexin 43) expresses in preim-
plantation embryos and involves in cell compaction [6]
and development [29]. In the current study, the expres-
sion of these gene transcripts was relatively quantified in
the d 8 blastocysts derived from 3 - 10 or <3 mm folli-
cles and cultured with serum or growth factor supple-
A. Dhali et al. / Open Journal of Animal Sciences 1 (2011) 97-105
Copyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/OJAS/
103
(a)
(b)
Figure 2. Variations (mean ± SEM) in the total number of cells
(panel A) and DNA fragmentation index (panel B) in bovine
blastocysts produced from the vitrified morulae. Morulae were
derived from 3 - 10 mm follicles following IVM and IVF in
serum-free media and IVC in serum (FBS, 4%) or growth fac-
tor (EGF, 10 ng/mL; IGF-1, 100 ng/mL; SCF, 50 ng/mL) sup-
plemented medium. Post-thaw culturing was done in the re-
spective medium for 4 d. a-b on error bar indicates a signifi-
cant difference (P < 0.05).
mentation. The expression of the transcripts varied sig-
nificantly in all cases, except for BIRC5 in the blasto-
cysts derived from 3 - 10 mm follicles. The variations in
the expression of these transcripts in the growth factor
treated embryos were found to be within a short range of
0.5 to 1.7 fold compared to that of the embryos treated
with FBS. However, from the observed profiles of gene
expression, it was difficult to interpret whether the qual-
ity of the growth factor treated embryos was better or
inferior compared to that of the embryos treated with FBS.
The ability of an embryo to withstand freezing and
thawing is a useful indicator of quality [5]. Apoptosis of
embryonic cells indicates an inadequate in vitro culture
environment and/or embryo freezing [25,30,31]. Altered
apoptosis levels in blastocysts may lead to early embry-
onic death or congenital anomalies in the fetus [32]. In
the current experiment, cryotolerance was assessed of
the embryos that were derived from 3-10 mm follicles
and were cultured in the presence of serum or combina-
tions of growth factors. In the Experiment-1, the sup-
plementation of the growth factors either individually or
in combinations resulted in similar embryo development.
Therefore, the combinations of growth factors were only
selected for this experiment assuming that supplementa-
tion of such combinations could be beneficial over the
individual growth factor supplementation for cryotoler-
ance of embryos. The embryos were vitrified at the
morula stage using the droplet vitrification method. We
have shown previously that this method is efficient for
preserving mouse embryos at the morula stage [33].
Following thaw and culture, the expanded/hatched blas-
tocyst rate was significantly greater when embryos were
treated with serum compared to growth factors. The re-
sults indicated better cryotolerance of the serum treated
embryos compared to those treated with growth factors.
Nevertheless, cryotolerance of the growth factor treated
embryos was not different as was evident from the simi-
lar survival, blastocyst and expanded/hatched blastocyst
rates of these embryos. There were no differences in the
total number of cells and DNA fragmentation index in
blastocysts derived from the vitrified morulae, indicating
a similar level of apoptosis in the embryos treated with
serum or growth factors.
In conclusion, embryo development was not different
among the culture medium supplementations irrespec-
tive of follicle size. Nevertheless, FBS and SCF supple-
mentations could not support satisfactory development
of the embryos derived from <3 mm follicles. The ex-
pression patterns of the development and apoptosis re-
lated gene transcripts in blastocysts indicated that quality
of the embryos treated with growth factors was different
compared to that of the embryos treated with serum.
Better cryotolerance was observed when embryos were
treated with serum compared to growth factors. Never-
theless, irrespective of culture medium supplementations,
a similar apoptosis level was evident in blastocysts de-
rived from the vitrified morulae.
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