Procedures for In Vitro Production of Bovine Embryos

Procedures for In Vitro Production of Bovine Embryos

The procedures for in vitro production [IVP; i.e. in vitro maturation (IVM), in vitro fertilization (IVF), and in vitro culture (IVC)] of embryos described here are based on procedures developed in other laboratories at the University of Wisconsin ( Parrish et al., 1986 ), University of Guelph ( Xu et al., 1992 ), and University of Missouri ( Hernandez-Ledezma et al., 1993 ). These procedures as used by our group have been published previously ( Edwards et al., 1997; Paula-Lopes et al., 1998 ). Keep in mind that the protocols described here are not fixed but rather constantly evolve as new developments take place. Therefore, practitioners of IVP will be well advised to experiment with the procedures used, especially after reading of improvements made by other laboratories. This protocol is organized by day of the protocol with d 0 being the day of fertilization.

DAY -2
PREPARATION FOR IN VITRO PRODUCTION OF EMBRYOS

Materials and Equipment Needed

2 x 1 L sterile saline (transport saline)
1 aliquot of stock 16 (Pen-Strep 10 ml) per bottle of saline
3 to 4 bottles of oocyte collection medium (OCM) (400 ml)
1 aliquot of stock 11 (glutamine 4 ml) per bottle of OCM
1 aliquot of stock 15 (Pen/Strep 4 ml) per bottle of OCM
1 aliquot of stock 4 (BSS + Hep 8 ml) per bottle of OCM
1 aliquot of oocyte maturation medium (OMM) (87 ml)
1 aliquot of stock 8 (gentamicin 1 ml) per aliquot of OMM
1 aliquot of stock 3 (BSS 10 ml) per aliquot of OMM
1 aliquot of stock 6 (Folltropin 125 ml) per aliquot of OMM
1 aliquot of stock 10 (glutamine 1 ml) per aliquot of OMM
200 ml of Stock 5 (estradiol) per aliquot of OMM
1 ml of Stock 2 (sodium pyruvate) per aliquot of OMM
Laminar flow hood

Procedure

1. The day before the collection of ovaries, prepare and place at 4°C until use.
a) Saline + Pen/Strep -add 1 aliquot (10 ml) of Stock 16: pen/strep to each liter of transport saline
b) OCM + Supplements - add supplements to OCM as described
c) OMM + Supplements - add supplements to OMM as described

2. Place saline (0.5 L/container) into two clean containers (Fig. 1). Place both containers in a thermos and leave at room temperature for use the next day.

Figure 1. This container is an example of one that is suitable for transportation of ovaries. Almost any leak-proof plastic container will work.

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DAY -1
RETRIEVAL OF OVARIES AND
OOCYTE COLLECTION AND MATURATION

OVARY COLLECTION

Materials and Equipment Needed

Thermos with 2 containers with 0.5 L of saline + pen/strep in each.
Appropriate attire as required by the slaughterhouse
Scalpels and scissors
Gloves

Procedure

1. Remove ovaries from the reproductive tract of cows immediately after internal organs are extracted from the carcass and place the ovaries into one of the saline containers.

2. After all ovaries have been collected, remove the excess blood from the ovaries by massaging the ovaries in the container. Then, transfer ovaries to the second container and place the containers back in the thermos.

3. Transport the ovaries to the lab immediately.

OOCYTE COLLECTION

Materials and Equipment Needed

Preparation of Microdrops
Incubator (5% CO2 and 38.5°C)
Laminar flow hood
100 ml pipet tips and pipettor
OMM + supplements
Mineral oil
60 x 15 petri dishes
Plastic pasteur pipet
3-4 bottles of OCM + supplement (warmed @ 38.5°C)
1 L saline + Pen/Strep (warmed @ 38.5°C)

Preparation for Oocyte Collection
Scalpel
Petri dishes with OMM + supplement microdrops (pre-equilibrated)
Bench paper to cover surface
25 ml pipet
automatic pipettor
Mesh or styrofoam on counter
X-plate
Integrid petri-dish100 μm Falcon Cell Strainer50-ml Centrifuge Tubes, self standing with plug seal cap (Fisher)Scalpel blades (#11 and #20)
Gloves
400 ml sterile beaker
Container to discard ovaries
Timer
slide warmer @ 38.5°C
Dissecting microscope
Water-bath at 38.5°C

Figure 2. Some culture plates needed for IVP. From left to right are a Falcon 60 x 15 petri dish used for preparing microdrops, a Nunclon 4 well plate for fertilization, a Fisher X-plate used in several steps for cleaning up preparations of COCs and embryos, and a Fisher intergrid plate for searching for cumulus-oocyte complexes. All can be purchased from Fisher .

Procedure

1. Place bottles of supplemented OCM and 1 L of saline at 38.5°C at least 4 h before ovaries arrive.
If the person doing oocyte collection is also going to the slaughterhouse to collect the tissue, the OMM
microdrops should be prepared and the saline and OCM be put in the oven before leaving. Otherwise,
OMM plates should be prepared ~ 1h before the expected time of ovary arrival.

2. At least two hours before they are needed, prepare several (60 x 15) plates containing nine, 50 ml microdrops (Figure 3) of OMM. Prepare enough OMM microdrops (10 oocytes/microdrop) to mature the number of oocytes expected to be collected. Cover the microdrops with mineral oil.

Figure 3. Preparation of microdrops. Shown here are 9-50 ml drops on the bottom of a 60 mm petri dish. To complete the drops, they are covered with mineral oil by depositing oil using a Pastuer pipette.

Figure 4. Some items needed for oocyte collection. Shown on the warming plate are 2 1-L plastic beakers (one containing cleaned ovaries and one to collect ovaries after processing) and a 400 ml beaker containing ~100 ml OCM. Also shown are a hemostat, scalpel handle, disposable scalpel blades and bench paper.

3. Upon return to the lab, wash ovaries (by massaging; Figure 5) several times with the pre-warmed saline until the majority of the blood has been washed away from the ovaries. Following the washes, place ovaries in the beaker containing fresh saline and store at 38.5°C until time of oocyte collection.

Figure 5. Massaging the ovary to remove blood.

4. Add ~100 ml oocyte collection medium (OCM) to a sterile 400 ml beaker.

5. Attach a hemostat to the base of the ovary to hold the ovary firmly in place. Cut the excess tissue from the ovarian stalk. Hold ovary above beaker and make 2 � 3 mm deep incisions across all visible follicles. See Figure 6 for illustration.

Both follicular fluid and blood in the collection medium could result in clotting of the medium , thereby rendering it impossible to retrieve oocytes. To prevent clotting of the medium, do not collect from large follicles (>10 mm). Either do not slash these follicles or rupture them before harvesting other follicles and discard the follicular fluid. Also, do not make incisions across corpora lutea. It is imperative not to cut too deep into the surface of the ovary (practice will aid with this) to avoid cutting larger blood vessels in the ovary.

Figure 6. Slashing of ovaries to recover oocytes. In panel A, a hemostat is attached to the base of the ovary to hold the ovary firmly in place. The excess tissue from the ovarian stalk is removed in panels B. Panels B and C shows how the ovary is held above the beaker and 2 � 3 mm incisions made in a a downward direction with a rapid but firm movement across follicles.

6. Submerge ovary into OCM and swirl vigorously. Repeat this process until the desired number of ovaries has been processed (See Figure 7). One can anticipate a yield of about 10 usable oocytes/ovary. Sometimes as many as 20-30 can be obtained).

Figure 7. Harvesting of oocytes from slashed ovaries. In the left panel, the slashed ovary is being swirled in OCM. In the right panel, the ovary is being pressed against the side of the beaker to allow drainage of OCM.

Figure 8. Cleaning up the preparation of oocytes collected by ovary slashing. Panel A depicts the placement of tubes in the water bath for settling and Panel B the supplies necessary for this step. Aspiration of oocytes from the bottom of the tube is shown in Panel C while Panel D represents the technique used to pour the aspirated pellet trhough the cell strainer. Rinsing of oocytes from the cell strainer into anintegrid petri dish is shown in Panel E.

7. After slashing ovaries, the medium containing the oocytes is poured into sterile 50-ml centrifuge tubes. Be careful not to overfill the tube which can cause oocyte loss. A good measurement is the 45-ml line.

Note: The number of tubes needed for the collection of oocytes depends upon the amount of medium you begin with in your 400-ml beaker after you are done slashing ovaries.

8. Place the tubes containing the oocytes and media into a water bath and allow oocytes to settle to the bottom of the centrifuge tubes for about 5 minutes (figure 8A).

9. While oocytes are settling, fill a 10 ml syringe with warmed OCM (for later use in rinsing the oocytes from filter) and pour ~ 2 ml OCM into an integrid petri-dish to prevent oocytes from sticking to the bottom of the plate.

10. Use a forceps to hold a 100 μm cell strainer in position over a sterile 100 ml beaker. Using a plastic pasteur pipet and sterile technique, aspirate the pellet of oocytes at the bottom of each tube (figure 8C). Slowly pour the aspirated pellet into the cell strainer (figure 8D). Up to 3 tubes of oocytes can be processed through a single filter. Note: If the strainer starts to clog, rinse some OCM through the filter to clear debris and make it easier to search for oocytes later on.

11. Immediately turn the filter upside down and, with a 10 ml syringe fitted with an 18g needle filled with OCM, rinse the oocytes into an integrid petri-dish (figure 8E). Place the intergrid dish on plate warmer until ready for searching (Figure 9).

Note: If it is necessary to process more than 3 tubes of oocytes, the cell strainer can be reused after completing step 11.

Note: There is an alternative protocol available for processing oocytes that does not involve use of the cell strainer. This is the original protocol used by us.

Figure 9. Searching for oocytes. After COCs have been washed, the contents are poured into an Intergrid plate and examined under a dissecting scope for COCs. These can be picked up using a wiretrol or other device.

12. Collect oocyte cumulus complexes (COC) as fast as possible to prevent adverse effects of cold shock. Only COCs which have at least a couple of layers of compact cumulus cells and an evenly granulated cytoplasm with no clear spaces should be used for subsequent steps. Place retrieved COCs into the first well of the X-plate containing OCM that was prepared in step 8 (Figure 10). Continue washing by transferring COCs between wells using a wiretrol pipet, microdispensor pipet, or the instrument of your choice to handle oocytes (Figure 11).

13. Transfer oocytes from one well to the next leaving all debris behind (repeat twice to assure that oocytes are clean of debris; Figure 10).

Figure 10. An X-plate used to wash COCs, purchased from Fisher .

Figure 11. Washing of COCs using an X-plate.

Figure 12. Some instruments used to pick up embryos and oocytes. From left to right are 1) a 1 cc syringe with an extension of rubber tubing connected to a Unopette , 2) a wiretrol (from Drummond Scientific; we purchase from Fisher ), 3) the same device as #1 without the rubber tubing extension and 4) a 5 ml Drummond Microdispensor (also purchased from Fisher ). For tips on how to use these instruments, click here .

14. After oocytes have been cleaned of debris, transfer groups of 10 to a 50 ml microdrop of OMM (Fig. 13). Note: It is essential that oocytes be collected, washed and incubated in OMM as quickly as possible to ensure maximum development rates.

Figure 13. Transfer of COCs to microdrops.

13. Incubate for 18 to 24 h at 38.5°C and 5% CO2 .

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Media Preparation for Day 0

1. The following media should be prepared on day -1 so that they are ready for the next day (Day 0)
HEPES-TL and HEPES-TALP
IVF-TL and IVF-TALP
Sp-TL and Sp-TALP
90% Percoll

2. Instructions for preparing media are available on the Media Preparation Page.

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DAY 0
IN VITRO FERTILIZATION

INITIAL PREPARATION FOR SPERM PURIFICATION AND FERTILIZATION

Materials and Equipment Needed

Laminar flow hood
90% Percoll
IVF-TALP
HEPES-TALP
Sp-TALP
Sp-TL
7 x 15 ml conical centrifuge tubes
3 centrifuge carriers
4 well plates
Thawing unit (Citothaw)
PHE
Pipet tips (1000 ml)and pipettor
Sterile pipets (1 x 5 ml and 1 x 2 ml)
Plastic Pasteur pipet

Procedures

The following procedures are done on the morning of day 0 (or a minimum of 2-3 hours before fertilization) so that all supplies and media are ready when fertilization procedures are initialized.

1. Fill a total of 4-5 15 ml conical tubes with HEPES-TALP. Tighten the caps and place in the incubator.
It may seem like 4-5 tubes is a lot but some of these tubes of HEPES-TALP will be used later in the day.

2. Add 10 ml Sp-TALP to a 15 ml conical tube. Tighten the cap and place in the incubator.

3. Add 5 ml IVF-TALP to a 15 ml conical tube. Leave cap loose and place in the incubator.

4. Prepare enough 4-well plates (Figure 2) for the number of oocytes that are maturing (30 oocytes/well). Add 600 ml of IVF-TALP to each well and allow medium to equilibrate and warm up for 2 h.

5. Place 1.5 ml of 90% Percoll and 1.5 ml of Sp-TL to one 15 ml conical tube. Mix to make a solution of 45% Percoll. In another 15 ml conical tube, add 3 ml of 90% Percoll. Make a Percoll gradient (45% over 90%) by slowly layering the 45% Percoll over the 90% Percoll by the use a plastic Pasteur pipet. Cap and place in the incubator.

6. Plug-in citothaw (i.e., thawing unit) so the water warms up.

7. Place 1-2 aliquots of PHE in the oven (25 ml per well) (remember to cover the tube with aluminum foil).

8. Place 2-3 centrifuge carriers in the oven.

PREPARATION OF OOCYTES FOR FERTILIZATION

Materials and Equipment Needed

X-Plate
HEPES-TALP (pre-warmed)
IVF-TALP (pre-warmed and equilibrated in CO2)
Sp-TALP (pre-warmed)
Percoll gradient (pre-warmed)
Dissecting microscope
Inverted microscope
60 x 15 petri-dish
Rack for tubes (place in front of the heater)
Heater
Scissors (wipe with ethanol)
Semen straw plunger (wipe with ethanol)
Light microscope
Plastic sterile Pasteur pipets
Pipettor (25 ml)
Pipet tips
Instrument to pick-up oocytes
Slide warmer (set at 38.5°C)

Procedure

1. Place X-plate on the slide warmer and add ~5 ml of HEPES-TALP to each of the wells.

2. Remove one or two dishes containing matured oocytes and place on the slide warmer.

3. Transfer COCs from each microdrop of OMM + supplement to the X-plate containing HEPES-TALP.
For ease of handling of oocytes and to speed up this step, transfer the contents of 3 microdrops (30 matured oocytes) into each corner of the X plate. Repeat as necessary until all oocytes have been placed in the corners of the X-plate in groups of 30.

4. Withdraw 4-well plate (containing pre-equilibrated IVF-TALP (600 ml/well) from the incubator and transfer a group of 30 oocytes from a corner of the X-plate to a well of a 4-well plate.

5. Return plate with the oocytes to the incubator until fertilization.

SPERM PURIFICATION USING PERCOLL

Materials and Equipment Needed
The materials and equipment for Preparation of Oocytes is also used for sperm purification

Procedure

Note: It is critical that spermatozoa not be exposed to cold shock. A space heater in front of the area where the sperm work will be performed can aid in preventing cold shock to the sperm cells (make sure you don’t roast the sperm by keeping it too close to the heater). Also, make sure that all media used for sperm are warmed to 38.5°C before use. Media necessary for fertilization should be prepared at least 2 h prior to IVF (HEPES-TALP, Sp-TALP, IVF-TALP, 90% Percoll).

1. Thaw 2-3 straws of semen in the citothaw for 45-60 seconds.
An alternative way to thaw semen straws is to place straws in a beaker of warm tap water (37° C). Note also that it is usually not necessary to use 2-3 straws. One straw provides enough semen for 4 wells (100-120 oocytes). Unless the choice of sire is critical, we typically pool semen from 2-3 bulls (1 straw per bull) to enhance the probability that sperm from at least one sire will perform well.

Figure 14. Transfer of straws of semen from liquid nitrogen tank to the thawing unit (citothaw).

2. Wipe the straw dry with a kimwipe, cut the tip of the straw with a scissors and expel contents of the straw onto the top of the Percoll gradient (Figure 15). Care must be taken so that the gradient is not disturbed and the semen lie on top of the 45% layer.
To facilitate removal of the semen, a homemade plunger can be devised to fit into the straw. Care should be taken not to push the cotton plug into the gradient.

Figure 15. Layering of sperm onto Percoll. After cutting the tip of the straw (Left panel), the contents of the straw are expelled onto the top of the Percoll gradient (right panel). Here, removal of the semen is facilitated by using a homemade plunger.

3. Place the conical tube containing the semen and Percoll gradient into a centrifuge carrier that has been pre-warmed to 38.5°C, and centrifuge at 1000 x g for 10 min.

4. After centrifugation, collect sperm pellet from the bottom of the conical tube (Figure 16).
Percoll is toxic to sperm cells and the pellet should be collected with a minimum of Percoll.

Figure 16. Removal of sperm from the bottom of the Percoll gradient.

5. Place the sperm pellet into a 15 ml conical tube containing 10 ml Sp-TALP and place in a warm centrifuge carrier before centrifuging for 5 min at 200 x g. The exact speed is probably not critical - do a low-speed centrifugation.

6. Remove the supernatant with a Pasteur pipet while being careful not to disturb the pellet (Figure 17). This step must be done quickly because motile sperm will swim out of the pellet. If the pellet is accidentally disturbed, stop the procedure and re-centrifuge.

Figure 17. Washing sperm in Sp-TALP. The left panel shows the washed and centrifuged sperm. The right panel shows the pellet of sperm remaining in the tube after aspiration of the supernatant.

7. Determine dilution required to add ~1-3 x 105 in 25 ml (4-7 x 106 /ml). To do so, add 10 ml sperm suspension to 90 ml water to kill sperm. Load 10 ml of sample onto a hemacytometer. Count the number of sperm in 5 squares (Figure 18) and multiply sperm number by 500,000 to determine concentration per ml. Dilute the sperm using IVF-TALP that has been pre-equilibrated in the incubator.
Alternatively, add ~ 0.5-1.0 ml of pre-equilibrated IVF-TALP to the sperm pellet (the bigger the pellet, the larger the amount of IVF-TALP to add to the pellet) and look at the concentration of sperm cells until it appears to be ~ 4-7 x 106 /ml (possible with practice).

Figure 18. Hemacytometer used for counting sperm. The total number of sperm in five of the smaller boxes (outlined by freehand) are counted and multiplied by 500,000 to determine concentration per ml. For more details on how to use a hemacytometer, click here .

FERTILIZATION

Materials and Equipment Needed

slide warmer (set at 38.5°C)
purified sperm
PHE

Procedure

1. Remove plates containing matured oocytes from the incubator and place on the slide warmer.

2. Add 25 ml sperm preparation and 25 ml PHE mix into each well.
When pipetting the sperm, place the pipette in the middle of the sperm suspension rather than on the bottom to avoid grabbing debris that can settle to the bottom of the tube .

3. Return 4-well plate to incubator for 8-10 h. Many people do fertilization for 18-20 h. When we were establishing IVF in our lab, 8-10 h gave better results than longer incubation times. Recently, however, we have gotten good results with 18-20 h fertilization times. In addition, longer fertilization times make it easier to remove cumulus cells after fertilization.
To determine the incidence of parthenogenesis, one well should be prepared without sperm, but with PHE. After 8 � 10 h, place these oocytes into a separate culture medium drop and culture for 2 days before looking at rate of parthenogenesis.

PREPARATION OF EMBRYO CULTURE DROPS

Materials and Equipment

KSOM
60 x 15 mm petri dishes
mineral oil

Procedure

1. Prepare embryo culture medium (modified KSOM) at least 2 h before removing embryos/oocytes from the fertilization drops.

2. Make enough 50 ml microdrops of culture medium (30 oocytes/embryos per drop) on 60 x 15 mm petri dishes and cover with mineral oil. We also prepare 25 m l microdrops when we wish to culture fewer numbers of embryo. Typically, we place 25-30 embryos in a 50 m l microdrop and 5-10 embryos in a 25 m l microdrop.

3. Place the dishes in the incubator to warm up and equilibrate.

TRANSFER OF FERTILIZED OOCYTES INTO EMBRYO CULTURE DROPS
(8-10 h post IVF)

Materials and Equipment Needed
Vortexer
Timer
1.5 ml Dolphin microcentrifuge tube and rack
X-plate (with prewarmed HEPES-TALP)
Plastic Pasteur pipet
Heater (placed in front of the microscope)
Slide warmer (set at 38.5°C)
Dissecting microscope
Instrument to pick-up embryos

Procedure

1. Rinse a 1.5 ml microcentrifuge tube with HEPES-TALP and leave a small (~50 ml) volume of HEPES-TALP in it.

2. Place X-plate on the slide warmer and add ~5 ml of HEPES-TALP to each of the wells.

3. After microscope and air have been warmed sufficiently, remove one 4-well plate containing IVF drops from the incubator.

4. Remove oocyte-cumulus complexes (now called putative zygotes since many of them have been fertilized) from each well of the 4-well plate and place in the microcentrifuge tube. Up to 300 embryos can be loaded in one microcentrifuge tube.

5. Repeat steps 3 and 4 until all plates have been processed.

6. Remove cumulus cells from embryos/oocytes by vortexing (Figure 18) the tube containing the embryos/oocytes for 5 min.
--A good technique is to press the tube hard so that the fluid is propelled to the top of the tube. Then rapidly, take the tube off the vortexer and repeat (i.e., kind of bounce the tube on the vortexer).
--If removal of all cumulus cells is imperative, vortex in the presence of hyalurodinase. Click here to view the protocol for cumulus removal using hyalurodinase.

Figure 18. Vortexing COCs to remove cumulus cells.

7. Transfer the putative zygotes from the tube to the X-plate and rinse tube 2-3 times with HEPES-TALP to gather all embryos.

8. Wash embryos/oocytes 2-3 time by transferring them from one well to the next in order to clean them of cells and debris.

Notes on steps 7-8: To avoid overflow, leave well #1 empty, place HEPES-TALP in wells #2 and #4 and embryo culture medium (KSOM) in well #3. Add embryos/oocytes to well #1, and rinse the centrifuge tube 2-3 times with HEPES-TALP from well 4. Remove all bubbles with the pipette to aid in visualization of the embryos and place the bubbles in well 4 (because embryos sometimes get stuck in the bubbles). Transfer embryos sequentially from well 1 to wells 2, 4 and 3.

9. Finally, transfer the putative zygotes to microdrops of pre-equilibrated culture medium [i.e. modified KSOM (KSOM-BE) or CR1aa]. We typically add 30 embryos/oocytes per microdrop but embryos can be cultured at other densities. We often prepare 25 m l microdrops when we wish to 5-10 embryos per drop .

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<center><a name="oocytes_into_culture_drops"></a><a name="top"></a>DAY 1-9 <br />EMBRYO CULTURE<p></p><p></p><p></p><p></p><p></p></center><center><a name="top"></a><a name="day 3"></a>DAY 3 AFTER IVF - ASSESSING CLEAVAGE RATE</center>

1. Pre-warm stage of inverted microscope by placing heater near the microscope for at least 15 min prior to use.

2. Assess cleavage rate of embryos by determining the number of embryos cleaved divided by the number of embryos/oocytes placed initially in the microdrops. Return plates to the incubator.

<center><a name="day 3"></a><p><a name="day 3"></a><a name="day 5"></a>DAY 5 AFTER IVF - ADDITION OF SERUM (OPTIONAL)</p><a name="day 5"></a></center>

1. Place aliquots of heat-inactivated fetal calf serum ( stock 24 ) in the incubator ~1-2 h before adding to the microdrops containing the embryos/oocytes.

2. Pre-warm air by placing the heater in front of the slide warmer.

3. Place plates on the slide warmer and add 5 ml of sterile filtered heat treated fetal calf serum to each microdrop and return to incubator.

<center><a name="day 5"></a><p><a name="day 5"></a><a name="day 7-9"></a>DAYS 7-9 AFTER IVF - ASSESSMENT OF DEVELOPMENT</p><a name="day 7-9"></a></center>

1. Pre-warm stage of inverted microscope by placing heater near the microscope for at least 15
min prior to use.

2. Assess development of embryos to the blastocyst stage. Return plates to the incubator.

Click here for description of embryos at different stages.

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ABBREVIATED IVP PROTOCOL

This checklist can be used to ensure that each step in the IVP procedure is completed successfully.

DAY �2

Preparation of Media
- saline
- OMM
- OCM

Check incubators for accuracy of temperature and CO2 readings

DAY -1

Collection of ovaries from slaughterhouse

Preparation for oocyte collection
- Place supplemented OCM and saline in the oven
- Prepare plates of OMM microdrops and cover them with mineral oil
- 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 200 ml of OCM to beaker
- Slice ovaries
- Swirl ovaries in the beaker
- Place beaker in water-bath
- Add OCM to beaker until full

Collection of oocytes-cumulus complexes from beaker (5 min. sedimentation time)
- Pipet off supernatant
- Add more OCM
- Place beaker in water bath
- Repeat until medium is clear

Collection of oocyte-cumulus complexes from beaker
- 25 ml pipet
- automatic pipettor
- mesh or Styrofoam on counter
- water-bath set at 38.5°C

Searching for oocyte-cumulus complexes
- X-plate
- Grid plate
- Dissecting microscope
- Searching instrument (microdispensor, wiretrol, etc)
- Slide warmer

-Decant in a grid plate.
-Transfer oocyte-cumulus complexes to X-plate and rinse 2X .

Oocyte maturation
- After the last rinsing place cleaned oocytes-cumulus complexes (10/drop) into a 50 ml microdrop of
pre-equilibrated OMM
- Mature oocyte-cumulus 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

DAY 0

Preparation of media for fertilization (~2.5 h prior to fertilization)
- Percoll gradient (loosen cap and place at 38.5°C)
3 ml 45% (1:1with Sperm-TL) over 3 ml 90%
-HEPES-TALP (tight cap and place at 38.5°C)
3-centrifuge tubes with 15 ml HEPES-TALP
- IVF-TALP (leave cap loose and place at 38.5°C)
4-well plates (600 ml/well)
1-centrifuge tube with ~5 ml IVF-TALP
- Sp-TALP (tight cap and place at 38.5°C)
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 OMM plate to a corner of an X-Plate containing HEPES-TALP. Repeat until all oocytes have been placed in the X-plate.
- 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 15 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.

DAY 3

Pre-warm stage of inverted microscope and room

Determine cleavage rate (be quick)

DAY 5 (OPTIONAL)

Place FBS tube in the incubator for 1-2 h prior to use

Pre-warm room

Add 5 m l FBS to each embryo containing microdrop (optional)

DAY 7-9

Pre-warm stage of inverted microscope and room

Collect data on blastocyst development

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DISHWASHING AND AUTOCLAVING

Soap Solutions

Soap bath (to wash glassware, and instruments)

Dilute PCC concentrate in deionized water according to manufacturer's instructions

7X Cleaning solution (ICN) � fill a squirt bottle for ease of use

Dishwashing
Dirty dishes are rinsed in water, any tape immediately removed, and soaked in soap bath until dishes are done. Do not leave dirty dishes to air dry and do not leave them with traces of medium inside.

Glassware, caps, collection medium carboy, stir bars, and instruments

1) remove any tape/label left on the bottle/caps (this should have been done beforehand)
2) fill bottle/beaker with some dilute PCC and shake
3) clean with brush
4) rinse 5X with warm/hot tap water (make sure there is no soap left)
5) rinse 5X with deionized water
6) rinse 1X with distilled/deionized water
7) Let air dry

Plastic beakers/containers

1) squirt some 7X cleaning solution into the container
2) wash with brush or sponge
3) rinse 5X with warm/hot tap water (make sure there is no soap left)
4) rinse 5X with deionized water
5) rinse 1X with distilled/deionized water
6) Let air dry

Autoclaving
after dishes are completely dried

Bottles/ collection medium carboy
place cap loosely onto the bottle and put a piece of autoclave tape.

Stir bars/ instruments
place inside Tower DualPeel ® autoclave tubing and tape at both ends. Place a piece of autoclave tape on the package.

Beakers/volumetric flasks
Cover snugly with a double layer of aluminum foil. Place a piece of autoclave tape on the foil.

Autoclave for a minimum of 25 minutes. After autoclaving is done, tighten bottle caps a little bit to prevent any contaminants to enter. Do not tighten caps completely until bottles have cooled to prevent a vacuum forming in the bottle.

STOCK SOLUTIONS

For ease of use, prepare aliquots of all solutions and keep frozen. Aliquots may be placed in a Styrofoam rack and the rack labeled with stock number, solution name, aliquot volume, and date.

The choice of water for making stocks depends upon local availability of highly-purified water. We make up stock solutions using Tissue Culture Water purchased from Sigma . For all other media (OCM, saline, etc.), we use Millique water or distilled and deionized water.

For product catalog number and company information, click on the highlighted word.

Stock 1:Na lactate. Purchase as a 60% syrup . Follow manufacturer’s indications for expiration date. Store at 4°C.

Stock 2: Na pyruvate. Dissolve 0.220 g sodium pyruvate in 100 ml water. Sterile-filter into an aluminum-foil wrapped 100 ml bottle and store at 4°C for 1 mo.

Stock 3: Bovine Steer Serum (BSS). Prepare 10 ml aliquots of BSS in sterile 13 ml tubes and store at -20°C indefinitely.

Stock 4: BSS/Hep. Add 1000 USP units of sterile heparin (dissolved in 3-5 ml of water and sterilize through a syringe filter) into 500 ml BSS (Stock 3). Store in 8 ml aliquots in sterile 13 ml tubes indefinitely at -20°C.

Stock 5: Estradiol. Dissolve 1 to 3 mg estradiol in ethanol for a final concentration of 1 mg/ml. Store in a glass container at -20°C for up to 2 months.

Stock 6: Folltropin. Reconstitute Folltropin-V as directed by manufacturer to prepare a 20 mg/ml solution. Place 150 ml aliquots into sterile 1.5 ml microcentrifuge tubes and store indefinitely at -20°C.

Stock 7: Heparin . Dissolve 20 mg in 10 ml water. Pipet 300 ml aliquots and store at -20°C in 1.5 ml microcentrifuge tubes indefinitely.

Stock 8: Gentamicin. Dilute to 5 mg/ml concentration with water and sterile filter. Pipet 1 ml aliquots into sterile 4 ml tubes and store at -20°C indefinitely.

Stock 8A: Gentamicin. When preparing Stock 8, prepare a few extra tubes of 10 ml aliquots in sterile microcentrifuge tubes and store at -20°C indefinitely.

Stock 9: PHE Mix. Prepare primary stocks of 1 mM hypotaurine (1.09 mg in 10 ml saline), 2 mM penicillamine (3 mg in 10 ml saline) and 250 mM epinephrine [1.83 mg in 40 ml of a solution prepared by mixing 77 ml of a 98% Na lactate syrup (or the equivalent volume if a lower percent lactate syrup is used], 50 mg Na metabisulfite and 50 ml water]. Epinephrine is easily oxidized by direct light so take precautions to avoid this problem (wrap in aluminum foil or place in dark container). Combine 5 ml of 1 mM hypotaurine, 5 ml of 2 mM penicillamine, 2 ml of 250 mM epinephrine and 8 ml of saline and sterile filter. Aliquot 400 ml of PHE Mix into sterile 1.5 ml microcentrifuge tubes and store in a light resistant container at -20°C indefinitely. Upon retrieval of PHE mix for use, wrap tube in aluminum foil.

Stock 10: Glutamine (1ml) . Prepare stock solution of 1.5 g glutamine /100 ml water, sterile filter and make 1 ml aliquots in sterile 4 ml tubes and store at -20°C indefinitely.

Stock 11: Glutamine (4 ml). Prepare stock solution of 1.5 g glutamine /100 ml water, sterile filter and store 4 ml aliquots in 13 ml tubes at -20°C indefinitely.

Stock 12: MgCl2 for Percoll. Prepare 0.1 M stock by adding 0.203 g MgCl2 to 10 ml water. Sterile filter and store at 4°C indefinitely.

Stock 13: CaCl2 for Percoll. Prepare 1 M stock by adding 0.735 g CaCl2 +2H2 O to 5 ml water. Sterile filter and store at 4°C indefinitely.

Stock 14: Hyaluronidase. Prepare stock solution of type IV hyaluronidase at 10,000 units/ml in saline, sterilize through a 0.2 mm filter into a sterile tube, and prepare 100 ml aliquots in sterile microcentrifuge tubes and store at -20°C indefinitely.

Stock 15: Pen/Strep (4 ml). Thaw 100 ml bottle of pen/strep and aliquot 4 ml into sterile 5 ml tubes and store at -20°C indefinetely.

Stock 16: Pen/Strep (10 ml). Thaw 100 ml bottle of pen/strep and aliquot 10 ml into sterile 13 ml tubes and store at -20°C indefinetely.

Stock 24: Fetal calf serum. Prepare 100 ml aliquots of heat-inactivated fetal calf serum in sterile microcentrifuge tubes and store at -20°C

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PREPARATION OF MEDIA

Transport Saline (0.9%)
Prepare 0.9% saline (9 g NaCl per liter) in double distilled water and autoclave. Label as "Sterile 0.9% saline, date made" and store indefinitely at 4°C.

Oocyte Collection Medium (OCM)
1. Dissolve TCM-199 powder (without phenol red, and without glutamine) (Atlanta Biologicals) for 10 L and 3.50 g NaHCO3 in 9 L ddH2 O. Adjust pH to 7.2-7.4 and bring volume to 10 L. Sterile-filter 400 ml medium into 500 ml bottles and keep indefinitely at 4°C. Labels should read "Oocyte Collection Medium, - Supplements and date made".

2. On night before use, add the following: 1 aliquot of stock 4: BSS+Hep, 1 aliquot of stock 11 : glutamine (4 ml), and 1 aliquot of stock 15: Pen/Strep (4 ml). Change label to "+supplements", change date and use within a week.

Oocyte Maturation Medium
1. Prepare 87 ml aliquots of TCM-199 . and store at 4°C until use.

2. On night before use, add the following to an aliquot of TCM-199:
1 aliquot stock 3 : BSS
1 aliquot stock 8 : gentamicin
125 ml stock 6 : Folltropin
200 ml stock 5 : estradiol
1 ml stock 2 : Na Pyruvate
1 ml stock 10 : Glutamine

Change label to read "Oocyte maturation medium" and use within 1 week.

TL Solutions - For Making TALPs
Note that these media can also be purchased from Cell and Molecular Technologies or Biowhittaker . Cell and Molecular Technologies maintain websites for ordering information for TL solutions and instructions for media preparation

1. To prepare media, mix the ingredients as described in Table 1 (all volumes are in milliliters), adjust the pH, check osmolarity (if osmometer is available) and sterile-filter the solution.

2. Write expiration date on the label (use within one week) and store at 4°C.

Table 1. Recipes for preparation of TL solutions

TALP (Tyrode’s Albumin Lactate Pyruvate) Media
1. To prepare media, mix the ingredients as described in Table 2 and sterile-filter the solution.

2. Write expiration date on the label (use within one week) and store at 4°C.

Table 2. Recipes for TALP Media