The Art and Practice of

Dr Osamu Dochi
Dr Jaswant Singh
Dr Carl Lessard
Dr Pritpal Malhi

Oocyte Competence Laboratory

Department of Veterinary Biomedical Sciences, Western College of Veterinary Medicine

52 Campus Drive, Saskatoon Sk S7N 5B4 Canada

2006-2007

1
 Table of Content
Day to Day Procedures
Day 1: IVM
Day 2: IVF
Day 3: IVC
Laboratory/Preparation Procedures
Transportation of Slaughterhouse Ovaries
Sperm Counting Procedure

2
 Day to Day Procedures
Day 1: IVM

Media Preparation and IVM Dish Incubation

1. Prepare IVM media in the morning (9AM) or min. 2 hours before receiving ovaries
2. Place 2x5ml vials of calf serum on warming plate for thawing (Takes 15 min.)
3. Place the following in the biosafety hood: 1x100ml sterile measuring cylinder,
2xFalcon culture bottles, 1x100ml sterile glass bottle and lid, 2x20ml all-plastic
syringe, 2x0.22µm syringe filter, TCM199 media, FSH vial, LH vial, gentamysin
vial, 1ml, 100µl and 10µl pipette tips and micropipettes.
4. Prepare 50ml IVM Media (TCM199+calf serum+hormones) as per IVM media
preparation instruction sheet
5. Prepare COC holding media (dPBS+3-5% calf serum), filter into glass bottle and
leave on warming plate for 2 hrs before use. Store at 40C
6. Prepare IVM dishes: Pipette out 2-3ml IVM media in a 35mm Falcon petri dish
for use, mark Drop1 position and label 35mm Falcon petri dishes, place 4x5µl
drops in each dish without touching the bottom of plate, cover with 2ml mineral
oil, add 95µl media to each drop and finally add 3ml (approximate) more mineral
oil to cover the drops completely. Place 2 dishes each in the cover of 90mm dish
7. Place the IVM dishes and IVM media in the 5% CO2 incubator

Ovary Collection
1. Collect ovaries at the slaughter house in a plastic Ziploc bag. Wash the ovaries
immediately with normal saline x2 at 220C. Drain completely
2. Transport ovaries at 20-220C in an insulated cooler

Follicle Aspiration and IVM

1. Check and record the temperature of ovaries on arrival in the laboratory
3. Wash ovaries with normal saline, trim off ovarian pedicle.
4. Rinse 5 ml syringe with 19G needle with PBS or follicular fluid.
5. Aspirate follicles and collect aspirate in a 50 ml falcon tube.
6. Take aspirate to the lab.
7. Scratch 7mm squares on the bottom of one large petridish (90 mm) for searching.
8. Prepare three glass pipettes (one for aspirating sediment + other two pulled for
COC pick up).
9. Add 2ml COC holding media in each 35mm petri dishes x3 (for COC washing)
and place on warming plate
10. Take sediment of follicle aspirate in your large dish (3 times).
11. Search and pick up COC and wash them three times in COC holding medium.
12. Three washings in IVM medium (2ml each) using 35mm petri dishes. Spread all
COC in a line in the final dish for easy pickup of different grades of COC.
13. Tansfer 20 COC into each 100µl IVM drop (5µl per oocyte; you can also use a
single 500µl drop for 100 COC) for oocyte maturation for 20-22 hours. Return
dishes as soon as possible to the 5% CO2 incubator
2007.02.12 (Pritpal/Jaswant)

3
 Day to Day Procedures
Day 2: IVF

Media Preparation and IVF Drop Preparation

1. Prepare media in the morning (9AM or at least 2 hours before start of IVF
2.

In Vitro Fertilization Procedure

1. Take a 5 ml sterile tube for semen and keep it at warm plate.
3. Take a 5ml pipette and 1 ml pipette and keep them on warm plate.
4. Take water in a container to thaw semen straws (Temp 37-38 C).
5. Take semen straws quickly and thaw them in water for 45 sec.
6. Cut semen straw(s) and pour semen into tube.
7. Get Percoll tube from incubator and slowly layer semen on the top (along the
wall; use 1 ml pipetter with sterilized tip).
8. Centrifuge at 2000 rpm for 20 min.
9. Prepare three glass pipettes (one for aspirating supernatant + other two pulled for
COC pick up).
10. Take small Petri dishes for oocyte washing (2 to 3 washings in oocyte wash
medium and maintain oocyte in last dish and put them back into incubator).
11. After centrifugation for 20 min, slowly aspirate supernatant leaving about 1 ml at
the bottom.
12. Get sperm washing solution from Co2 incubator.
13. Add 6 ml of sperm washing sol. to sperm pellet (use 5 ml pipette, Warm your
pipette and mix after adding).
14. Centrifuge at 2000 rpm for 5 min.
15. Take 5 ml of saline solution for sperm counting in a tube, discard 50 µL out of it
(use 1 ml pipetter with sterilized tip).
16. Keep your counting chamber ready with cover slip on it.
17. After centrifugation, aspirate supernatant leaving 0.8 ml in the tube.
18. Take 50 µL of sperm suspension after thorough mixing, and mix it with 4950 µL
of saline solution.
19. Measure rest of the sperm suspension with 1 ml pipette and transfer it back to the
incubator.
20. Vortex tube for counting sperms (10 times on vortex and 30 times with hand).
21. Take 50 µL from the middle and load counting chambers.
22. Count sperms (say 10 sperms i.e. 10 million/ml).
23. Calculate [10 X volume of sperm suspension /6] – volume of sperm suspension =
required volume of sperm washing sol to adjust sperm conc to 6 million per ml
24. Adjust conc to 6 million per ml using sperm washing sol from incubator (say vol
is 1.3ml).
25. Adjust conc. to 3 million per ml using dilution medium from incubator (add 1.3
ml of diluting solution).
26. Add 95 µL of sperms to 5 µL of IVF drops.
27. Add oocytes (20 oocytes per drop).
28. Transfer your dish to the incubator.

4
 Laboratory/Preparation Procedures
Transportation of Slaughterhouse Ovaries

At the Oocyte Competence Laboratory

1. Clean the container with wet paper towels immediately on arrival. Let it dry.
2. Replace the absorbent pad. Place 1 litre normal saline bag and a large ziploc
freezer bag. Store the data logger in the battery compartment.
3. Plug in the power supply of the container for battery charging (charger is in
the container).
4. Ship the container to Moosejaw Veterinary Clinics through Relay Courier
(306-____ in Saskatoon) at least 2 day before the next collection date.

At the Moosejaw Veterinary Clinics (306-692-3622)

1. Plug in the power supply of the container for battery charging (charger is in
the container).
2. Check that the power switch is off (charging will continue even when the
container is switched off).
3. Unplug the charger and store in the container just before going to slaughter
house.

At the Slaughter House

4. Turn on the power switch of container and confirm that temperature is set at
240C. Turn on the power switch of the temperature logger (red switch on the
left side) and hold down the REC/STOP button to start recording (Red button)
till the “REC” appears on the LCD screen.
5. Collect ovaries in the plastic bag.
6. Wash the ovaries twice using about 500 ml of saline at 200C each time.
7. Drain completely and place the ziploc bag in plastic box.
8. Place the channel #1 temperature sensor of the temperature logger in the
middle of ovaries.
9. Place the temperature recorder in the charger compartment and place the
channel # 2 temperature sensor outside the container (please insert the sensor
between the container wall and black carrying box).
10. Send the ovaries to Saskatoon via Relay Courier Inc. (306-631-6295 in
Moosejaw). Please advice courier driver to deliver ovaries first on arrival in
Saskatoon.

On arrival at the Oocyte Competence Laboratory

1. Switch off the temperature data logger and download the data to the computer
at later time.
2. Record temperature of the ovaries with a mercury/digital thermometer.
3. Wash the ovaries two or three times with a total of 1 litre normal saline.
4. Trim off ovarian pedicle and extra tissues.
2007.02.01 (Dochi/Singh)

5
 Laboratory/Preparation Procedures
Sperm Counting Procedure

Calculations
1. The hemocytometer is 0.1 mm deep and the 25 large squares represent an area of
1 square mm. The volume in 25 squares is 0.1 mm3 = 0.1 µl.
2. Sperms counted in 0.1 µl X 10,000 = sperms counted in 1 ml or 1000 µl
3. We diluted 50 µl of sperm suspension to 5 ml or 5000 µl i.e. 100 times dilution.
4. Conc. of sperms/ml = number of sperms in 0.1 µl X 10,000 X 100 (dilution
factor). That is, conc. of sperms = (counts from 25 large squares) millions/ml

Procedure

6
 Chemicals and Supplies
Chemicals and Reagents
Reagents Conc. Amount Supplier Cat # Storage Expiry & Comments
MP Biomedicals
7X Laboratory Detergent (ICN7667094) VWR Room Temp.
No.:7667094
0
Albumin, Bovine V 50g SIGMA A-8022 4C
Albumin, Bovine V 50g SIGMA A-9418 40C
Albumin, Bovine (crystallized and
25g SIGMA A4378
lyophillzed)
Albumin, Bovine(Essentially Fatty Acid
10g SIGMA A-6003 40C
Free)
Albumin, Bovine(Fatty Acid Free: low
5g SIGMA A-8806 40C
endotoxin)
BME amino acids solution 50x 100ml SIGMA B6766 40C
Calcium chloride(CaCl2 2H2O) 500g SIGMA C-7902 Room Temp.
Calf serum ?????? -200C
Delbecco’s Phosphate Buffer Saline 1000 ml 21300-
Invitrogen Room Temp.
(with Ca++ and Mg+) x10 025
Delbecco’s Phosphate Buffer Saline
500 ml Invitrogen Room Temp.
(with Ca++ and Mg+)
Delbecco’s Phosphate Buffer Saline
500 ml Invitrogen Room Temp.
(without Ca++ and Mg+)
Embryo surfactant Bioniche Room Temp.
Ethylene glycol 1000ml SIGMA E9129
Fetal calf serum ?????? -200C
FSH (Folltropin) Bioniche Room Temp. Expiry printed on vial
Gentamicin sulfate 1g SIGMA G-1264 40C
Hepalean 1000 unit/ml 10ml SIGMA 40C
Organon
Heparin(solution) 1000unit/ml 10ml Room Temp.
Teknika

7
Reagents Conc. Amount Supplier Cat # Storage Expiry & Comments
Heparin 10,000unit SIGMA H-3149 Room Temp.
HEPES 25g SIGMA H-6147 Room Temp.
Hypotaurine 100mg SIGMA H-1384 Room Temp.
(+)-Lactic acid (Hemicalucim
50g SIGMA L-4388 40C
salt:Hydrate)
Lactic acid 100ml SIGMA L-1375 40C
Lactic acid Sodium salt) 100ml SIGMA L-4263 40C
L-Glutamin(C5H10N2O3) 25g G-8540 Room Temp.
L-Glutamin(C5H10N2O3) 100g G-1517 Room Temp.
LH (Lutropin) Bioniche Room Temp. Expiry printed on vial
Magnesium Chloride(MgCl2) 100g SIGMA M-8266 Room Temp.
Magnesium Chloride(MgCl2 6H2O,
100g SIGMA M-2393 Room Temp.
hexahydrate)
12340-
Medium 199 500ml GIBCO 40C
030
Medium 199 (HEPES modification) 150.6g SIGMA M-2520 40C
MEM amino acid solution 50x 100ml GIBCO 1113-051 40C
MEM amino acid solution 50x 100ml SIGMA M5550 40C
MEM Non-essential amino acid solution 100x 100ml SIGMA M-7145 40C
MEM Non-essential amino acid solution 100x 100ml SIGMA M-7146 40C
11140-
MEM Non-essential amino acid solution 100x 100ml GIBCO 40C
050
2-mercaptoethanol 100ml SIGMA M7522
Methyl 4-hydroxybenzoate 100g SIGMA H-6654
161403-
Mineral oil 1000ml Aldrich
1L
Percoll 500ml SIGMA P-1644 40C
Percoll 100ml SIGMA P-1645 40C
Phenole Red 0.5% in 100ml SIGMA P-0290 40C

8
Reagents Conc. Amount Supplier Cat # Storage Expiry & Comments
DPBS
Phenole Red 5g SIGMA P-3532 Room Temp.
Phenole Red 5g SIGMA P-5530 Room Temp.
Potassium chloride(KCl)) 50 SIGMA P-5405 Room Temp.
Pyruvic acid 25g SIGMA P-5280 40C
Sodium Bicarbonate(NaHCO3) 500g SIGMA S-5761 Room Temp.
Sodium Bicarbonate(NaHCO4)??????? 500g SIGMA S-4019 Room Temp.
Sodium chloride(Nacl) 1kg SIGMA S-5886 Room Temp.
Sodium chloride(Nacl, SigmaUltra) 250g SIGMA S-7653 Room Temp.
Sodium Phosphate (NaH2PO4 H2O,
250g SIGMA S-9638 Room Temp.
Anhydrous) ???????
Sodium Phosphate
100g SIGMA S-5011 Room Temp.
Monobasic(NaH2PO4, Anhydrous)
Sucrose 1kg SIGMA S1888 Room Temp
GIBCO/ 12340-
TCM-199 500ml
Invitrogen 030
Water for embryo transfer 500ml SIGMA w1503 4C
50g SIGMA S-1277 4C

9
 Chemicals and Supplies
Plasticware and Glassware
Item Quantity Catlog # Supplier Comments
Disposable centrifiuge tubes Case of 500 21008-929 VWR(Falcon)
Absorbent bench underpads (deluxe) 10x30 56617-018 VWR 17x24inch
Filter 12/case 28198-505 VWR(Nalgene)
Filter 12/case 28199-677 VWR(Nalgene)
Filter 250mL 157-0020 Nalgene
Millex GP filter Unite (0.22um) 1 case SLGP033RS Millipore
Millipore Sterivex filter unit 15 z35,991-2
Petri Dish 10/500 153066 Nunclon
Petri Dish 10/400 150288 Nunclon
Petri Dish 627102 Greiner bio-one
Petri Dish 35mm 35mm 1003 FALCON
Petri Dish 60mm 60mm 1007 FALCON
Spatula Pack of 3 57952-107 VWR
Storage bottle 250ml 430281 CORNING
Straw (0.25ml) 2000 5565 IMV
Vacuum filtration system (Corning) 1 case F9893
Weighing paper Pack of 500 12578-121 VWR

10
 Media Preparations
2006.10.15

Preparation of In Vitro Maturation Medium (TCM199)

Without*
With Hormones Hormones
Preparation volume 10 mℓ 20 mℓ 30 mℓ 40 mℓ 50 mℓ 100 mℓ
(mℓ)
TCM 199 (mℓ) 9.5 19 28.5 38 47.5 95
Calf Serum (mℓ) 0.5 1 1.5 2 2.5 5
FSH solution (μℓ) 2.50 5 7.5 10 12.5 -
LH solution (μℓ) 10 20 30 40 50 -
Gentamycin 10 20 30 40 50 100
solution (μℓ)

* You may prepare the Maturation Media for use over two day, but you must add the
hormones on the day of use. Maturation Media without hormones should be filtered
and stored at 4○C.

- Thaw a vial of Calf Serum (takes about 15 minutes)

- Use 50 or 100 ml sterile measuring cylinder to prepare the solution in Biosafety
hood
- Pour in approximate volume of TCM 199 media in the cylinder and add last 3 to 4
ml with 1ml micropipette to adjust the volume accurately
- Add all remaining solutions in order (as listed above) using micropipettes
- Cover with Parafilm and mix gently by inverting the cylinder few times
- Filter into a Tissue Culture Flask using All-plastic 20ml syringe and 0.22µ
syringe filter
- Close the lid, loosen it one turn, and store the media in CO2 incubator for at least
2 hrs before use

11
 July 24, 2006
By Dochi
Preparation of CR1aa
1) Preparation of Stock solution
Preparation volume (mℓ) 760 mℓ 500 mℓ 250 mℓ 100 mℓ
Sodium chloride : NaCl (g) 6.7031g 4.4099g 2.2050g 0.8820g
Potassium chloride : KCl (g) 0.2311g 0.1520g 0.0760g 0.0304g
Sodium pyruvate (g) 0.0440g 0.02895g 0.0145g 0.0058g
Sodium bicarbonate : NaHCO3 (g) 2.2011g 1.4481g 0.7240g 0.2896g
0.5% Fenol Red (mℓ) 2 mℓ 1.3158 mℓ 0.6579 mℓ 0.2632 mℓ
HEPES(g) 0.4529g 0.2979g 0.1490g 0.0596g
Note; storage in refrigerator less than 1 month

CR1 Stock B
Preparation volume (mℓ) 200 mℓ 40 mℓ 20 mℓ
L(+)-Lactic acid Hemicalcium Salt (g) 0.5996 0.11992 0.05996
Note; storage in refrigerator less than 1 month

L-Glutamic acid solution

Preparation volume (mℓ) 30 mℓ
L-Glutamic acid (mg) 60mg
Note; storage in refrigerator less than 1 month

12
 Jun 20, 2006
By Dochi
2) Preparation of CR1aa
CR1aa
Preparation volume (mℓ) 100 mℓ 50 mℓ
Solution A (mℓ) 76 mℓ 38 mℓ
Solution B (mℓ) 20 mℓ 10 mℓ
BME Essential Amino Acids (x50) (mℓ) 2 mℓ 1 mℓ
MEM Nonessential Amino Acids (x100) (mℓ) 1 mℓ 0.5 mℓ
L-Glutamic acid (mℓ) 1 mℓ 0.5 mℓ
Bovine Serum Albumin (Fatty Acid-free) (g) 0.3g 0.15 g
Gentamaicn solution (μℓ） 100μℓ 50μℓ
Note; storage in refrigerator less than 1 week

ＣＲ１aa + 5 % CS
Preparation volume (mℓ) 30 mℓ
CR1aa (mℓ) 28.5 mℓ
CS (mℓ) 1.5 mℓ
Note; storage in refrigerator less than 1 week

3) Preparation of culture drop for IVC

 20 to 25 oocytes / 30μℓ
 ５％ CO2, 5% O2, 38.5℃

13
 Jun 20, 2006
By Dochi
Ｐｒｅｐａｒａｔｉｏｎ ｏｆ ＢＯ solution

BO Stock solution A
Preparation volume (mℓ) 500 mℓ
Sodium chloride : NaCl (g) 4.3092g
Potassium chloride : KCl (g) 0.1974g
Calcium chloride dihydrate: CaCl2・2H2O(g) 0.2171g
Sodium dihydrogen phosphate dihydrate: NaH2PO4・2H2O(g) 0.084g
Magnesium chloride hexahydrate: MgCl2・6H2O(g) 0.0697g
0.5% Phenol Red (μℓ） 100μℓ
Note; storage in refrigerator less than 1 month

BO Stock solution B
Preparation volume (mℓ) 200mℓ
Sodium bicarbonate: NaHCO3(g) 2.5873g
0.5% Phenol Red (μℓ） 40μℓ
Note; storage in refrigerator less than 1 month

14
BO solution
Preparation volume (mℓ) 100mℓ 50 mℓ
Stock solution A (mℓ) 76mℓ 38mℓ
Stock solution B (mℓ) 24mℓ 12mℓ
Sodium pyruvate (g) 0.01375g 0.0069g
Gentamaicn solution (μℓ） 100μℓ 50μℓ

Sperm washing solution

Preparation volume (mℓ) 20mℓ 10mℓ
BO solution (mℓ) 20mℓ 10mℓ
Hyptaurin (g) 0.02182g 0.0109g
Heparin sodium solution (μℓ） 100μℓ 50μℓ

Oocyte washing solution

Preparation volume (mℓ) 30mℓ 20mℓ
BO solution (mℓ) 30mℓ 20mℓ
BSA(crystallized and lyophilized) (g) 0.3g 0.2g

Sperm diluting solution

Preparation volume (mℓ) 10mℓ
BO solution (mℓ) 10mℓ
BSA(crystallized and lyophilized) (g) 0.2g

15
Preparation of Percoll

10 x BO Stock solution A (for percoll solution)

Preparation volume (mℓ) 50 mℓ
Sodium chloride : NaCl (g) 4.3092g
Potassium chloride : KCl (g) 0.1974g
Calcium chloride dihydrate: CaCl2・2H2O(g) 0.2171g
Sodium dihydrogen phosphate dihydrate: NaH2PO4・2H2O(g) 0.084g
Magnesium chloride hexahydrate: MgCl2・6H2O(g) 0.0697g
0.5% Phenol Red (μℓ） 100μℓ

90% Percoll solution

Preparation volume (mℓ) 10 mℓ
Percoll solution (mℓ) 9mℓ
10 x BO Stock solution A (mℓ) 0.76mℓ
Sodium bicarbonate: NaHCO3(g) 0.03105g
H2O(mℓ) 0.24mℓ
Gentamaicn solution (μℓ） 10μℓ

45% Percoll solution

Preparation volume (mℓ) 10 mℓ
BO solution (mℓ) 5mℓ
90% Percoll solution(mℓ) 5mℓ

16
Preparation of Percoll gradient
1) Add 2 mℓ of 45% Percoll solution into 15 centrifuge tube.
2) Add 2 mℓ of 90% Percoll solution under the 45% Percoll solution.
3) Add semen on the 45% Percoll solution.
4) Centrifuge the tube at 2000 rpm for 20 min.
5) Aspirate out the supernatant and re-suspend the sperms in 6 mℓ of sperm washing
solution.
6) Centrifuge the tube at 1800 rpm for 5 min.
7) Aspirate out the supernatant then 50μℓ of sperm suspension for counting the sperm
number and measure the volume of sperm suspension.
8) Adjusting the sperm concentration at 6 X 106 using sperm washing solution.
9) Add an amount of sperm diluting solution equal to the volume of the sperm
suspension after the first dilution, to adjust at 3 X 106.
10) Add 95μℓ of the sperm suspension to each drop in the Petri dishes (having 5μℓ drop
of sperm diluting solution).

17
Preparation of Gentamaicin solution

① Gentamaicin 0.15g
② Physiological saline 3mL
③ Filter-sterilization using 0.22μm

18
FSH (Folltropin) preparation for IVM

Folltropin: 400mg FSH/ vial

1. Add 80 mL TCM 199 (or 0.9% Nacl salin) to Folltropin (5000μg/mL,

5μg/μL)
2. Mix the solution by inverting several times
3. Filter (0.22μm)-sterilization
4. Dispense into tube
5. Store frozen

Final concentration: 0.5μg/mL

Total IVM medium (mL) FSH solution
10 mL 10μL
30 mL 30μL
50 mL 50μL

19
 Aug 14, 2006
LH (Lutropin-V) preparation for IVM

1. Add 5 ml TCM199 to LH (Lutropin, Lot#:E092A)

2. Mix the solution by inverting the container several times
3. Filter-sterilization
4. Dispense into containers
5. Store frozen

At the time of using:

Stock solution: 5000μg/mL,
Final concentration: 5ug/mL
Total IVM medium (mL) LH solution
10 mL 10μL
30 mL 30μL
50 mL 50μL

20
 2006/12/05
Preparation of β-mercaptoethanol (BME) solution
for frozen-thawed embryos culture

β-mercaptoethanol: Sigma M7522 100mL

Preparation of BME stock solution

1. Place 10 ml of TCM199 in a test tube.
2. Remove 7 μℓ of TCM199 from the test tube by a micropipette.
3. Add 7 μℓ of BME.
4. Seal the test tube with Parafilm and mix contents by inverting several times.
5. Filter-sterilize and store in a refrigerator or a freezer (-20 C).

Preparation of TCM199 + 20% FCS + 0.1mM BME

1. Place 7.9 mL of TCM199 in a test tube.
2. Add 100μℓ of stock BME solution with a micropipette.
3. Add 2.0 mL FCS (fetal calf serum).
4. Add 10μℓ of gentamaicin stock solution.
5. Seal the test tube with Parafilm and mix contents by inverting several times.
6. Filter-sterilize

21