ABBREVIATED IVP
PROTOCOL
Day -2 Day -1 Day 0 Day 3 Day 5 Day 7-9
This checklist can
be used to ensure that each step in the IVP
procedure is completed successfully.
Preparation
of Media
- saline
-
- OCM
Check
incubators for accuracy of temperature and CO2 readings
Collection
of ovaries from slaughterhouse
Preparation
for oocyte collection
- Place supplemented OCM and
saline in the oven
- Prepare plates of
- Place plates in the
incubator
- Set up for oocyte collection
-
Scalpel
-
Scalpel blades
-
Gloves
- 400
ml beaker
-
container to discard ovaries
-
bench paper to cover surface
Harvesting oocyte-cumulus
complexes from ovaries
- Add 100 ml of OCM to beaker
- Slice ovaries
- Swirl ovaries in the beaker
-Pour OCM and oocyte
mixture into 50 ml sterile centrifuge tubes and place into water bath
Collection
of cumulus oocyte complexes (COCs)
from centrifuge tubes (5 min. sedimentation time)
- Set up for oocyte collection
- 100
mm cell strainer
- sterile
transfer pipets
- 10
ml airtite syringe
- 18
gauge needle
- Grid
plate
- Set up cell strainer over
50 ml beaker
- Using transfer pipet, suck up pellet and place in cell strainer
- Flip cell strainer over
into a grid plate on a slide warmer
- Fill needle/syringe with
OCM and rinse the debris from the strainer into the grid dish
Searching for cumulus oocyte complexes
-
X-plate
- Dissecting microscope
- Searching instrument (microdispensor,
wiretrol, etc.)
-
Slide warmer
-Transfer cumulus oocyte complexes to X-plate and rinse two times
Oocyte maturation
- After the last rinsing
place cleaned cumulus oocytes complexes (10/drop)
into a 50 ml microdrop of
pre-equilibrated
- Mature cumulus oocyte complexes for 18-24 h (place maturation plate in the
back of the incubator)
Prepare
media for fertilization
- HEPES-TALP
- IVF-TALP
- Sperm-TALP
- 90% Percoll
Preparation of media for fertilization (~2.5 h
prior to fertilization)
- Percoll
gradient (tighten cap and place in warm oven)
3 ml 45% (1:1with Sperm-TL) over 3 ml 90%
-HEPES-TALP (tighten cap and
place in warm oven)
3-centrifuge tubes with 15 ml HEPES-TALP
- IVF-TALP (leave cap loose
and place at 38.5°C in 5% CO2)
4-well plates (600 ml/well)
1-centrifuge tube with ~5 ml IVF-TALP
- Sp-TALP (tighten cap and
place in warm oven)
1-centrifuge tube with 10 ml Sp-TALP
- PHE (place in oven)
- Warm-up centrifuge
canisters (place in oven)
- Plug in citothaw
Matured oocytes: setup for washing and
fertilization
- X-Plate with HEPES-TALP
- Searching instrument
- Dissecting microscope
- Heater
- Scissors
- Semen straw plunger
- Inverted microscope
- Small petri-dish
- Rack for tubes (place in front of the heater)
- Slide warmer
- Plastic sterile Pasteur pipets
- Pipette (25 ml)
- Pipet tips
Matured oocytes: washing and
fertilization
- Transfer 3 groups of 10 oocytes-cumulus complexes from the
- Transfer the groups
of 30 matured oocytes-cumulus complexes to a well of
the 4-well plate containing
pre-equilibrated
IVF-TALP. Put plates back in the incubator.
Sperm
preparation (working in front of the heater)
- Place 1-3 straws of semen
in citothaw.
- Layer semen on top of Percoll gradient.
- Place Percoll
tube in a warmed centrifuge canister.
- Centrifuge for 10 min at
1000 x g.
- Collect semen pellet with a
Pasteur pipet.
- Place pellet into the 10 ml
Sp-TALP tube.
- Place Sp-TALP tube into a
warmed centrifuge canister.
- Centrifuge for 5 min at 200
x g.
- Pipet
off supernatant down to the pellet.
- Add IVF-TALP and determine
concentration.
Fertilization
- Add 25 ml of semen to well containing oocytes.
- Add 25 ml of PHE to each well.
- Place 4-well plates back in
the incubator and allow fertilization to proceed for 8-10 h (place plate in the
back
of the incubator).
Culture media
- KSOM
- Prepare plates with 50 ml microdrops of KSOM
and cover with mineral oil
- Place plates in the
incubator to equilibrate for at least 2 h
Setup for removal of oocytes/embryos
from fertilization drop
- Vortexer
- slide warmer
- Hyaluronidase (optional) –warm up
- Sterile dolphin-nose microcentrifuge
tubes (and holder)
- Heater (in front of microscope)
- X-Plate
- HEPES-TALP
- Dissecting microscope
- Searching instrument
- Timer
Remove oocytes/embryos from fertilization drop
- Rinse microcentrifuge
tube with HEPES-TALP and leave ~30-50 ml for collection of oocytes/embryos.
- Transfer oocytes/embryos from the fertilization drop to the microcentrifuge tube.
- Vortex the tube for 5 min
in front of the heater.
- Transfer the contents of
the microcentrifuge tube to a well of the X-plate
with a Pasteur pipet (rinse the
tube
several times).
- Search for cumulus-free oocytes.
- Wash 2X in HEPES-TALP.
- Transfer in groups of up to
30 to the KSOM microdrops.
- Place culture plate in the
back of the incubator.
Pre-warm stage of inverted microscope and room
Determine cleavage rate (be quick)
Place FBS tube in the incubator for 1-2 h prior
to use
Pre-warm room
Add 5 ml FBS
to each embryo containing microdrop (optional)
Pre-warm stage of inverted microscope and room
Collect data
on blastocyst development
this page was last updated
all original material for this
website © Rocio
Rivera, Peter J. Hansen et al.
2000-2002
Links to commercial sites do not constitute endorsement by the authors or the University of Florida
