Transcript Effect of energy source during culture on in vitro embryo

Effect of energy source during culture on in vitro embryo
development, resistance to cryopreservation and sex ratio
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Despite many efforts to improve embryo
culture systems in different species, the oviduct
remains irreplaceable for embryo
development.
Differences between in vivo and in vitro
produced embryos have been reported in
metabolic and morphologic profiles at the
cellular level [1,2,3], as well as in gene
expression [4,5], resistance to
cryopreservation [6] and post-transfer viability
[7]
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It is known that the environment has an impact on
the developmental physiology of the embryos. Of
the different steps of the in vitro production,
embryo culture is likely to have the major
influence, as embryos are held in media for up to
eight days.
It is also known that culture conditions influence
the sex ratio [9,10].
Indeed, another limiting factor of IVEP consists in
the alteration of the sex ratio towards males that
is often recorded in the in vitro system [11–13].
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Most of the embryo culture systems include glucose as
the main energy source.
Alterations of metabolic profile have been reported in
embryos cultured in the presence of glucose at blood
serum concentrations [16].
embryos can metabolize glucose from the zygote to the
blastocyst stage [2,3,17–19]; however, limited amounts
of glucose are oxidized by the tricarboxylic-acid cycle
whereas the major fate of the hexose is lactate
production [18,20]. The high lactate production
associated to glycolytic activity [2] has been reported to
reduce embryo development and viability [21].
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Interestingly, the presence of glucose during IVC
inhibits the development into blastocyst of
female embryos more than that of the male
counterparts [10], indicating a selective
embryotoxicity towards females.
In standard tissue culture medium (TCM)
glucose is present at the concentration found in
human serum, i.e., 5.6 mM [24]. whereas in
defined culture media, such as standard SOF
[25], the concentration of glucose is 1.5 mM.
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In a preliminary work, we demonstrated
that bovine blastocysts can be produced in a
medium containing minimum concentration
of glucose and enriched with glyceraldehyde
3-phosphate (G3P), an intermediate
metabolic compound of glycolysis, that
enters directly the pay-off phase of the
glycolytic pathway [32].
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Therefore, the aim of this work was to
evaluate whether minimizing the glucose
concentration in the medium or replacing
the hexose with other energy substrates
and/or embryotrophic compounds, in a
static culture system, would affect the in
vitro embryo development, the resistance to
cryopreservation and the sex ratio.
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compared 4 different culture systems:
1) 1.5 Mm glucose, as in standard SOF;
2) 0.15 mM glucose;
3) 0.125 mM G3P, in the presence of 0.15
mM glucose
4) 0.34 mM citrate, in combination with
2.77 mM myo-inositol.
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Reagents and media
The aspiration medium was TCM 199 supplemented
with 25 mM Hepes, 2 mM sodium bicarbonate, 2 mM
sodium pyruvate, 1 mM L-glutamine, 10 L/mL
amphotericin B (H199) supplemented with 2% bovine
serum (BS) and 95.6 SI/mL heparin.
The in vitro maturation (IVM) medium was TCM 199
supplemented with 15% bovine serum (BS), 0.5 g/mL
FSH, 5 g/mL LH, 0.8 mM L-glutamine and 50 g/mL
gentamycin.
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The IVF medium was Tyrode’s modified medium
[33] without glucose and bovine serum albumin
(BSA), supplemented with 5.3 SI/mL heparin, 30 M
penicillamine, 15 M hypotaurine, 1 M epinephrine
and 1% of BS.
The IVC medium consisted of Synthetic Oviduct
Fluid (SOF) medium [25], with 30 L/mL essential
amino acids, 10 L/mL non essential amino acids
and 5 % BS, that was supplemented with different
energy substrates in each experimental group, as
described in subsection 2.5.
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The base medium for the vitrification and
warming solutions was H199 with 20% fetal
calf serum (FCS).
The equilibration medium (VS1) consisted of
7.5% DMSO and 7.5% ethylene glycol (EG) in
base medium and the vitrification solution
(VS2) consisted of 16.5% DMSO and 16.5% EG
with 0.5M sucrose in the base medium.
The warming solutions consisted in 0.25 M and
0.15 M sucrose in the base medium.
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Cumulus-oocyte complexes (COCs) were
aspirated
Groups of 25 COCs were matured in 400 L of
IVM medium, covered with mineral oil, in four
well plates
COCs were washed and transferred, 25 per well,
into 300 L of IVF medium covered with mineral
oil.
The sperm were added in the fertilization wells
at the concentration of 1 106 sperm/mL.
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randomly distributed (30–50 per well, into 400
L of IVC medium) into 4 culture groups differing
from each other in the energy substrate.
After Incubation: On Day 7 (Day 0 IVF day)
cleavage and blastocyst rates were recorded
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The blastocysts produced were vitrified by the
Cryotop method
For warming, after removal of the cap, the
Cryotop strip was immersed directly into 1 mL of
the 0.25 M sucrose solution for 1 min and the
embryo was transferred into 200 L-droplet of
0.15 M sucrose solution for 5 min and then they
were washed.
Survival rates were evaluated.
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PCR products were analyzed using a 2% agarose
gel with 0.1 g Ml-1 of ethidium bromide in the
gel. The bands migrated by electrophoresis and
the products were observed with UV
transillumination.
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Following IVF presumptive zygotes (n 2625)
were randomly assigned to 4 experimental
groups in which the IVC medium was
supplemented with:
1.5 mM glucose (group A; n 851);
0.15 mM glucose (group B; n 554); 0.125 mM
G3P in the presence of
0.15 mM glucose (group C; n 608), and
0.34 mM tri-sodium citrate 2.77 mM myoinositol (group D; n 612).
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The efficacy of the different culture systems to
support embryo development was evaluated by
comparing the percentages of Grade 1 and 2
blastocysts (Bl) in relation to total COCs, as well
as the percentages of advanced embryos, i.e.,
expanded and hatched blastocysts (XBl and HBL,
respectively) out of the total blastocysts
produced, by the end of culture.
the survival and developmental rates of
blastocysts were evaluated after 24 h culture.
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Differences among groups in th percentages
of blastocysts, of advanced embryos out of
the total produced, as well as in the survival
rates after vitrification warming, were
analyzed by Chi square test.
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Differences in cleavage rates were observed among
groups, with the highest valu recorded in group B (P
0.05).
With regard to post-fertilization embryo development,
blastocyst rate was lower (P 0.01) in group A than in the
other three groups, that gave similar values.
However, the proportion of oocytes reaching the most
advanced stages of development (XBl and HBL) by the
end of culture decreased in group D (11.4%) compared
to both group A (15.8%; P 0.05) and groups B and C
(18.8 and 20.3, respectively; P 0.01).
The percentage of advanced embryos out of the total
blastocysts was also lower (P 0.05) in group B
compared to groups A and C
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the blastocysts produced in group D showed the
worst resistance to cryopreservation, as
indicated by the reduced (P 0.01) percentage of
embryo survival, evaluated on morphological
basis, after 24 h culture post-warming.
in group D the development rate, i.e., the
percentage of blastocyst reaching a more
advanced stage of development after in vitro
culture post-warming was lower (P 0.05) than in
group B, with groups A and C showing
intermediate values.
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The energy substrate affected the sex ratio of
the embryos produced.
a higher (P 0.05) percentage of female embryos
was obtained in group D (59/96 61.5%)
compared to group A (68/149 45.6%), whereas
intermediate values were found in groups B
(62/116 53.4%) and C (64/128 50%).
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The first finding of the present study was that
cleavage rate was significantly improved when
glucose concentration was reduced up to 0.15
mM, i.e., 1/10 of the standard concentration in
SOF (group B). Although sperm penetration has
already occurred by the time the presumptive
zygotes are transferred into IVC, it is likely that if
they encounter an adverse or suboptimal
environment the first cleavage division may be
impaired.
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In fact, both reducing glucose concentration in
the presence (group C) or absence of G3P (group
B) and replacing the hexose with citrate and
myo-inositol (group D) significantly improved
blastocyst production compared to standard SOF
containing 1.5 mM glucose (group A). Also the
percentage of cleaved oocytes developing into
blastocysts was reduced in group A compared to
the other groups.
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Culture conditions have a major influence on
survival an development of in vitro-produced
embryos following cryopreservation.
the slower kinetics of embryo development,
together with the poorer cryotolerance indicate a
reduced viability of the embryos cultured in
group D.
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It is known that in the in vitro system the sex
ratio is altered towards males and that male
embryos develop in vitro faster than female
embryos .
the sex ratio was altered towards males only in
the group with higher concentration of glucose
(group A), whereas an increased percentage of
female embryos was achieved in group D, i.e.,
the only system that did not include glucose,
apart for the very small amount present in the
BS.
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When glucose concentration was reduced, both in the
presence(group C) and absence (group B) of G3P, the sex
ratio was not skewed, with approximately 50% o
embryosof each gender.
supplementation of culture media with blood plasma-like
glucose concentrations causes a preferential loss of female
embryos during culture to the blastocyst stage.
It results that glucose exerts a selective embryotoxicity
towards female embryos at a concentration lower than 2
mM.
The higher vulnerability of female embryos to suboptimal
culture conditions may be due to the unbalanced
expression of X-linked genes before X inactivation occurs
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The higher vulnerability of female embryos to
suboptimal culture conditions may be due to the
unbalanced expression of X-linked genes before X
inactivation occurs.
Both the lower resistance to cryopreservation and
the slower development of the embryos produced in
group D may be related to the higher incidence of
female embryos. In fact, previous reports have
demonstrated that female embryos show a slower
developmental rate and are more sensitive to
cryopreservation.
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These results suggest that manipulating the
metabolic profile of the embryos during
culture may have an impact on sex ratio.