Taft1
Protocol
Project
protocol
—
Contents
Equipment
Common
reagents,
supplies,
solutions
Procedure:
superovulation
Procedure:
in
vitro
fertilization
Procedure:
embryo
freezing
and
thawing
Procedure:
embryo
transfer
Data
References
Definitions
COCs:
cumulus
oocytes
complexes
are
obtained
following
superovulation,
and
are
the
primary
source
of
mature
oocytes
for
in
vitro
fertilization
(IVF).
eCG/PMSG:
equine
chorionic
gonadotrophin/pregnant
mare
serum
gonadotrophin,
originates
in
the
endometrial
cups
and
detected
in
the
blood
during
pregnancy.
This
steroid
hormone
is
used
in
promoting
or
stimulating
the
growth
of
follicles,
and
used
in
combination
with
prostaglandins
to
promote
superovulation.
hCG:
human
chorionic
gonadotrophin,
is
a
steroid
hormone
with
characteristics
like
those
of
luteinizing
hormone
(LH).
It
is
produced
by
the
ovaries
and
secreted
in
the
placenta
during
gestation,
and
is
used
to
induce
ovulation
and
for
synchronization
of
estrus.
HTF:
is
a
synthetic
human
tubal
fluid
media,
and
is
used
for
in
vitro
fertilization
(IVF),
and
embryo
transfer
(ET).
M2:
is
a
chemically
synthesized
embryo
culture
media,
and
is
used
in
combination
with
cryoprotectant
propylene
glycol
(PG)
in
the
freezing
of
2-cell
embryos.
Workflow,
sampling,
and
experimental
treatment*
| Day |
Manipulation |
Phase |
Hormone
Rx |
Media/Tx |
1 |
Females
are
injected
i.p.
between
5-8
pm |
|
|
|
3 |
Females
are
induced
to
ovulate
mature
oocytes |
|
|
|
3 |
Oviducts
are
harvested |
Ovulation |
- |
HTF |
4 |
Caudal
epididymis
and
vas
deferens
are
harvested |
Sperm
collection |
- |
HTF,
light
mineral
oil |
4 |
Female
recipients
in
proestrus
are
mated
to
vasectomized
males |
Pseudopregnancy |
- |
7-13
fresh
2-cell
embryos
|
4 |
Pooled
COCs
are
added
with
5
µL
sperm |
|
- |
HTF |
4 |
Count
1-cell,
2-cell
embryos,
dead,
fragmented
oocytes |
|
- |
HTF |
5 |
Two-cell
embryos
are
pooled |
|
- |
HTF
first,
then
M2
and
cryoprotectant |
5 |
Females
with
vaginal
plugs
are
used
as
recipients |
|
- |
7-13
thawed
2-cell
embryos |
17-18 |
Uterus
is
harvested:
count
live/dead
fetuses
and
implantation
sites |
|
- |
|
*(Byers
&
Taft,
2005)
Equipment
- Bench-top
incubator
(Barnstead
International,
IA,
USA)
with
mixed
gases
(5%CO2,
5%O2,
balanced
with
N2),
with
constant
temperature
(37°C),
and
pH
(7.2-7.4)
- Modular
incubator
chamber
(Billups-Rothenberg
Inc.,
Del
Mar,
CA,
USA)
- Dissecting
microscope,
37°C
tissue-culture
incubator,
micro-dissecting
instruments.
Common
reagents,
supplies,
solutions
•
Human
tubal
fluid
(HTF)
medium
is
prepared
in
advance
(Sigma,
St.
Louis,
MO,
USA)
Synthetic
human
tubal
fluid
(HTF)
medium
recipe:
| Ingredients |
Components |
Millimolar |
Inorganic
salts |
Sodium
chloride
(NaCl) |
101.6 |
| Potassium
chloride
(KCl) |
4.69 |
| Magnesium
sulfate
(MgSO4⋅7H20) |
0.20 |
| Potassium
phosphate
(KH2PO4) |
0.37 |
| Calcium
chloride
(CaCl2⋅2H2O) |
2.04 |
| Sodium
bicarbonate
(NaHCO3) |
25 |
| Sugar
(energy
source) |
Glucose |
2.78 |
| Sodium
pyruvate |
0.33 |
Antioxidant |
Sodium
lactate |
21.4 |
Antibiotic |
Penicillin |
100
U/mL |
| Streptomycin |
50
µg/mL |
pH
indicator |
Phenol
red
(1%)
dye |
0.001%
(wt/vol) |
Protein |
BSA
(bovine
serum
albumin) |
4.0
mg/mL |
- Light
mineral
oil
(Fisher
Scientific,
USA)
is
washed
with
HTF.
- The
HTF
and
oil
are
allowed
to
stand
until
the
oil
is
separated
and
easily
decanted,
and
then
filtered
(0.8
mm
filter)
before
use.
- The
used
HTF
wash
is
discarded.
Common
to
all
procedures
Refer
to
Byers
&
Taft,
2005
for
precise
details
of
this
study.
a.
Oocyte
donor
females
are
17-27
days
of
age
and
sperm
donor
males
are
8-31
weeks
of
age.
b.
Hormonal
injections
are
delivered
intraperitoneally
(i.p.)
between
5-8
pm.
c.
Males
and
females
(13-14.5
hours
post-hormonal
treatment)
are
euthanized
by
cervical
dislocation.
d.
Female
CByB6F1/J
mice,
9
–
13
weeks
of
age
and
in
proestrus,
are
mated
to
vasectomized
CByB6F1/J
males,
to
induce
pseudopregnancy,
and
to
serve
as
recipients.
e.
Culture
dishes
and
medium
are
equilibrated
in
37°C
tissue-culture
incubator
30
min.
before
use.
f.
Microdissecting
instruments
are
autoclaved
and
sterilized
before
use.
Environmental
test
conditions
Date
of
experiment
is
accomplished
from
September
through
October.
Harvested
oocytes
are
scored
as
follows:
| Oocyte/embryo
score |
Description |
| Normal
or
Live
2-cell |
2-cell
with
intact
zonae
pelucida,
homogenous
cytoplasm,
and
morphologically
sound |
| Normal
or
Live
1-cell |
1-cell
with
intact
zonae
pelucida,
homogenous
cytoplasm,
and
morphologically
sound |
| Dead
|
Appear
dark
with
granular
cytoplasm |
| Fragmented |
Show
signs
of
blebbing
or
cellular
swelling,
resulting
in
breaks
and
fragmentation |
-----------
Superovulation
Purpose:
To
establish
baseline
performance
values
for
10
inbred
strains
following
hormonal
treatment
to
superovulate.
Reagents,
supplies,
solutions
- Equine
chorionic
gonadotrophin:
eCG
(PMSG;
CalBioChem
LaJolla,
CA,
USA)
- Human
chorionic
gonadotrophin
hCG
(Sigma,
CG-10
St.
Louis,
MO,
USA)
- Tissue
culture
dishes
Procedure:
superovulation
Inbred
strains
of
mice,
listed
as
high
priority
on
the
Mouse
Phenome
Database,
are
used
for
their
popularity
as
progenitors
for
recombinant
inbred,
consomic,
or
congenic
strains,
and
in
the
creation
of
genetically
modified
animals.
All
strains
(129S1/SvImJ,
A/J,
BALB/cJ,
BALB/cByJ,
C3H/HeJ,
C57BL/6J,
DBA/2J
FVB/NJ,
NOD/LtJ,
and
SJL/J)
are
subjected
to
the
same
procedures,
thus
allowing
comparisons
to
be
made
among
strains
on
the
number
of
eggs
produced
in
response
to
superovulation,
the
proportion
of
two-cell
embryos
produced
following
in
vitro
fertilization
(IVF),
and
the
proportion
of
live
pups
born
following
the
transfer
of
fresh
and
thawed
two-cell
embryo.
a.
On
day
1,
between
5-8
pm,
donor
females
are
first
treated
with
5.0
IU
of
eCG
(or
PMSG,
pregnant
mare
serum
gonadotrophin)
to
induce
follicular
development.
b.
46-50
hours
later,
on
day
3,
donor
females
are
given
5.0
IU
of
hCG
(human
chorionic
gonadotrophin)
to
induce
ovulation
of
mature
oocytes.
Within
approximately
12
hours
post-hCG
administration,
ovulation
occurs.
c.
Following
ovulation,
13-14.5
hours
after
the
last
hormone
treatment,
the
oviducts
are
harvested,
and
placed
in
culture
dish
with
300
µL
drop
of
fresh,
37°C,
human
tubal
fluid
(HTF)
medium.
d.
Using
a
dissecting
scope
and
microdissecting
forceps,
the
mass
of
oocyte/cumulus
complexes
(COCs)
are
released
from
the
ampulla
of
the
oviducts.
e.
The
cumulus
oocyte
complexes
are
pooled
so
that
2-6
COCs
(more
for
A/Js)
are
added
with
each
fertilization
drop
containing
sperms
(see
IVF
or
Byers
&
Taft,
2005).
Definitions
&
formulas
Percent
females
responding
to
superovulation
=
(#
females
responding
to
superovulation
÷
#
females
injected
to
superovulate)*100.
Total
oocytes
per
donor
=
(normal
live
oocytes
per
donor
+
dead
oocytes
per
donor
+
fragmented
oocytes
per
donor).
----------
In
Vitro
Fertilization
(IVF)
Purpose:
To
establish
baseline
performance
values
for
10
inbred
strains
following
in
vitro
fertilization
of
fresh
or
frozen
embryos.
Reagents,
supplies,
solutions
- IVF
culture
dishes
(60
mm
x
15
mm
standard
BD
Falcon
petri
dish
(Fisher
Scientific,
USA))
- Sperm
collection
dishes
(35
mm
x
10
mm
standard
BD
Falcon
petri
dish
(Fisher
Scientific,
USA))
Procedure:
in
vitro
fertilization
a.
IVF
tissue
culture
dish
is
prepared
with
the
addition
of
one
250µL
microdrop
fertilization
medium
(HTF),
two
150
µL
microdrops
of
wash
medium
(HTF),
and
one
150
µL
microdrop
of
culture
medium
(HTF),
all
covered
light
mineral
oil.
b.
Sperm
collection
culture
dish
is
prepared
with
a
1mL
microdrop
of
HTF
covered
with
a
layer
of
light
mineral
oil.
c.
Both
caudal
epididymides
and
vas
deferens
are
harvested
and
placed
in
the
sperm
collection
dish
prepared
above.
d.
The
epididymides
are
sectioned
several
times
and
then
incubated
for
10
min
to
facilitate
spermatozoa
motility
and
collection.
Sperm
motility
is
visually
assessed.
e.
A
5
µL
aliquot
of
epididymal
sperm
is
placed
in
an
IVF
culture
dish
using
a
wide-bore
pipette
tip
followed
by
the
addition
of
pooled
cumulus
oocyte
complexes
(see
superovulation
or
Byers
&
Taft,
2005),
and
then
incubated
for
4
hours
in
a
modular
incubator
chamber
at
37°C
in
5%
CO2,
5%
O2,
90%
N2.
During
this
fertilization
stage,
the
cumulus
cells
are
displaced
by
the
sperm;
thus,
facilitating
the
counting
of
normal,
fragmented,
and
dead
oocytes.
f.
Oocytes
appearing
healthy
are
washed
twice
with
two
150
µL
microdrops
of
HTF
to
remove
cellular
debris
(unbound
sperms
and
cumulus
cells)
and
then
incubated
overnight
in
a
third
150
µL
microdrops
HTF
culture
medium,
before
the
end
of
day
3.
g.
On
day
4,
about
18
hours
after
the
last
wash,
two-cell
embryos,
one-cell
embryos,
dead,
and
fragmented
oocytes
are
assessed
and
counted
(see
Oocyte
count).
Investigators
notes:
Two-cell
embryos
are
pooled,
so
that
around
35-65
embryos
per
IVF
dish
are
immediately
transferred
into
pseudopregnant
females
as
described
in
Embryo
transfer
or
Byers
&
Taft,
2005.
Excess
embryos
are
frozen
or
cryopreserved
as
described
in
Embryo
freezing
and
thawing
or
Byers
&
Taft,
2005.
---------
Embryo
Freezing
and
Thawing
Purpose:
To
establish
baseline
performance
values
for
10
inbred
strains
following
embryo
cryopreservation
and
embryo
thawing.
Equipment
- Controlled
rate
freezer
(Biotronics
DB1,
ZMS
Inc.,
FL,
USA)
- Liquid
nitrogen
and
liquid
nitrogen
container
(Thermo
Forma,
OH,
USA)
- Pulled
glass
pipettes
- Water
bath,
temperature
controlled
Reagents,
supplies,
solutions
- 1.0
M
sucrose,
1.5
M
propylene
glycol
(PG)
- 0.25-mL
plastic
insemination
straws
(IMV,
Maple
Grove,
MN,
USA)
- Critoseal
(Fisher
Scientific,
USA)
- M2
medium
(Glenister
&
Rall,
1999)
Procedure:
embryo
freezing
and
thawing
a.
To
freeze
two-cell
embryos,
the
embryos
are
first
pipetted
from
the
HTF
medium,
next
put
into
M2
medium,
and
then
finally
a
cryoprotectant
solution
of
M2
with
PG
for
15
min.
b.
Storage
straws
are
prepared
and
layered
with
3.5
cm
layer
of
sucrose
medium,
0.5
cm
air,
followed
by
0.7
cm
of
propylene
glycol,
and
then
2.0
cm
of
air
(See
Figures
1
and
2
below).
c.
Using
a
finely
tapered
pulled
glass
pipette,
about
30
embryos
are
loaded
in
the
propylene
glycol
layer
of
the
prepared
storage
straw
above
before
it
is
sealed
with
Critoseal.
d.
Embryos
in
straws
are
held
at
-7°C
for
at
least
5
min
before
being
seeded,
cooled
at
0.3°C
/min
using
a
controlled
rate
freezer
to
-30°C,
and
then
plunged
and
stored
in
liquid
nitrogen.
e.
Liquid
nitrogen
stored
frozen
embryos
are
thawed
from
straws
held
in
ambient
air
for
40
s,
and
then
quickly
placed
in
room
temperature
water
bath
for
15
s
to
rid
of
ice
(See
Figures
4
and
5
below).
f.
The
straw
is
blotted
dry,
before
the
Cristoseal
is
removed,
and
then
fitted
with
a
metal
plunger
used
to
extrude
the
thawed
embryos
into
a
culture
dish
(See
Figure
5
below).
g.
The
embryos
are
assessed
morphologically
and
scored
for
viability
(see
Oocyte
count);
after
5
min
they
are
placed
into
M2
medium
and
transferred
into
pseudopregnant
females
as
described
in
Embryo
transfer.
Investigator
notes:
The
number
of
embryos
frozen
for
each
strain
varied
depending
on
the
number
of
embryos
produced
during
the
IVF
procedure.
For
some
strains,
very
few
live
embryos
were
recovered
after
thawing
and
this
limited
the
number
of
embryo
transfers
performed
for
those
strains
(i.e.,
A/J,
BALB/cJ,
and
SJL/J).
Figure
1.
Figure
2.
Figure
3.
Figure
4.
Figure
5.
------------
Embryo
Transfer
Purpose:
To
establish
baseline
performance
values
for
10
inbred
strains
following
the
transfer
of
fresh
or
previously
frozen
embryos
into
pseudopregnant
recipient
females.
Procedure:
embryo
transfer
a.
Pseudopregnant
recipient
females
are
mated
(day
3)
and
then
examined
the
following
morning
(day
4)
for
vaginal
plugs.
b.
Only
females
with
vaginal
plugs
are
used
as
recipients
for
the
transfer
of
7-13
fresh
or
previously
frozen
(see
embryo
freezing
and
thawing)
embryos
into
one
oviduct.
c.
Embryo
transfer
is
accomplished
in
three
separate
days.
d.
Pregnant
embryo
recipients
are
fed
an
11%
fat
rodent
chow
(LabDiet1
5LA6,
St.
Louis,
MO,
USA)
throughout
gestation.
e.
By
day
17-18
of
pregnancy,
or
19-21
days
after
fertilization,
the
recipient
female
uterus
is
harvested
and
the
numbers
of
live
neonate,
implantation
sites,
and
resorbed
fetuses
are
recorded:
| Score |
Description |
| Live
neonate |
Neonates
are
able
to
move
and
breathe,
and
have
a
sanguine
color |
| Dead
|
Fetuses
appear
morphologically
normal,
but
never
moved
or
breathed |
| Implant
sites |
Sites
are
seen
as
small
dark
spots
on
the
uterine
wall
in
the
absence
of
an
embryo |
Figure
6.
Data
collected
by
investigator
- Superovulation:
total
oocytes
per
donor
- In
vitro
fertilization:
number
of
2-cell
and
1-cell
embryos,
dead
embryos,
and
fragmented
oocytes
- Fresh
embryo
transfer:
number
of
live
pups,
implant
sites,
and
resorbed
fetuses
- Frozen
embryo
transfer:
number
of
live
pups,
implant
sites,
and
resorbed
fetuses
-
Note:
Because
of
the
nature
of
these
data,
individual
data
set
rows
(and
the
N
associated
with
each
strain
mean)
can
represent
either
culture
dishes,
donors,
or
embryos
depending
on
context.
No
animal
ids
or
similar
case
identifiers
are
available.
|
|
only
Test age:
various
