RIKEN BRC protocols (2023-1)

IVF with frozen sperm
1.

Preparation of culture dishes
On the day of experiment, drops of medium are prepared as follows, and kept in a CO2 incubator(37 °C, 5% CO2 in air) to equilibrate for at least 20 min.
1)

For oocyte collection and fertilization: a 35 mm plastic dish (per four females) containing four80 L drops of 1.25 mM reduced glutathione (GSH) -mHTF medium* overlaid with oil (Fig. 1a).

2)

For sperm preincubation: a 35 mm plastic dish containing a 300 L drop of 0.4mM MBCD-PVA-HTF medium** (about 15 mm in diameter, with the edge widened with the tip of apipette) overlaid with mineral oil (Fig. 1b).

3)

For embryo culture: a 35 mm plastic dish containing twelve 10 L drops of CZB medium
containing glucose (or KSOM a/a medium) overlaid with mineral oil (Fig. 1c).

Fig. 1a2.

bc

Collection of oocytes
1)Collect mature MII oocytes from females who have been
induced to superovulate, and place them into the 80 Ldrops of GSH-mHTF medium (Fig. 1a).

3.

Thawing of frozen sperm1)

Retrieve a straw containing the frozen sperm from

Fig. 2
sperm solution

label

the liquid nitrogen with cryogloves and a face

PB1

mask to avoid accidental injury.
2)

After holding the straw in the air for 10 s, immerse
it in a water bath at 37 °C for 15 min (Fig. 2).

cutmetal stick4.

Preincubation of the recovered sperm
1)Fig. 3

Wipe the surface of the straw with a paper towel and expel the sperm solution onto the bottomof a plastic dish. The sperm solution takes up about 5 mm of the length of the straw and isenclosed in the end opposite the label. It is more opaque than the PB1 solution (Fig. 3).

2)

Cut the straw with scissors between the edge and the sperm solution, and retrieve only thesperm solution by pushing it out with a metal stick (plunger) inserted from the opposite end orwith a micropipette. Care must be taken not to mix the sperm solution with the PB1 solution,
which is also enclosed in the straw as a weight balancer.

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RIKEN BRC protocols (2023-1)
3)

Transfer the entire volume (~10 L) of sperm solution to the drop of 0.4mM MBCD-PVA-HTF
medium in the culture dish (Fig. 1b).

4)
4.

Incubate it (37 °C, 5% CO2) for 40-50 min.

Insemination
1)

After preincubation, check the condition of sperm, for example, hyperactivated movement,
concentration of sperm and percentage of motile sperm under the dissecting microscope.

2)

Add about 15 L of the thawed sperm suspension to a drop of GSH-mHTF medium containing
the oocytes. It is important to collect motile sperm from the periphery of a drop with a thin
pipette (Fig. 4). When the motility of sperm is less, or the number of oocytes are more than 40,
add about 10-20 L of the sperm suspension.

Fig. 4

Immortile
spermMotile
sperm3)Check the condition of the oocytes enclosed by cumulus cells after about 15 min frominsemination. If the amount of sperm solution in the drop of fertilization dish is not enough,add 10 - 20 l of sperm solution from sperm preincubation dish.

5.

Transfer the oocytes to the culture medium
1)

At 3–4 h after insemination, pick up the morphologically normal oocytes and wash them in two
drops of PB1 or M2 medium.

2)
6.

Transfer these oocytes into culture drops of fresh CZB or KSOM a/a medium (Fig. 1c).

Assessment of fertilization and embryo transfer
1)

On the day after IVF, count the numbers of two-cell embryos and the unfertilized oocytes,
respectively. We usually calculate the fertilization rate as: no. two-cell embryos/(no. two-cellembryos + morphologically normal unfertilized oocytes).

2)

The two-cell embryos are transferred into the oviducts (usually about 6-10 embryos/oviduct) ofpseudopregnant females on the day 0.5 (the day following sterile mating).
RIKEN Bioresource Research Center
Keiji Mochida
E-mail : keiji.mochida@riken.jp

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RIKEN BRC protocols (2023-1)

mHTF

PVA－HTF＊

mCZB

KSOM＊ ＊

Reagents

Campanies

Cat #SizemM

mg /100 ml

mg /100 ml

mg /100 ml

mg /100 ml

NaCl

FUJIFILM Wako Chemicals

191-01665

500 g

101.6

581.7

581.7 mg

476.5 mg

555 mgKCl

FUJIFILM Wako Chemicals

163-03545

500 g

4.7

3535

35.8

18.6

KH2PO4

FUJIFILM Wako Chemicals

169-04245

500 g

0.4

55

16.3

4.8

MgSO4・7H2O

FUJIFILM Wako Chemicals

131-00405

500 g

0.2

55

29.6

5

NaHCO3

FUJIFILM Wako Chemicals

191-01305

500 g

25210

210

211210

CaCl2・2H2O

FUJIFILM Wako Chemicals

033-25035

500 g

23030

25

25.1

EDTA・2Na

FUJIFILM Wako Chemicals

343-01861

50 g

---

3.7

0.4

Polyvinyl alcohol

Sigma-Aldrich

P8136

250 g

--

1010-

Phenol red sodium salt

Sigma-Aldrich

P4758

5g

-

0.2

0.2

0.2

0.2

GlucoseFUJIFILM Wako Chemicals

049-31165

500 g

2.8

50

50

100

3.6

Na pyruvate

Sigma-Aldrich

P8574

5g

0.3

3.6

Sodium DL-lactate (60% sylup)

Sigma-Aldrich

L7900100 mL

23.3

400 l3.6
400 l3.5370 l

2.2
174 l

L-Glutamine reduced

Sigma-Aldrich

G6013

5g---

14.6

14.6

Hypotaurine

Sigma-Aldrich

H1384

250 mg

-

1111-

-

Penicillin G・K salt

Sigma-Aldrich

P7794

1 MU

-

10106

6.3

Streptmycin sulfate

FUJIFILM Wako Chemicals

196-08511

5g---

55Albumin, Bovine Serum,

Merck Millipore

12657

25 g

-300

-300100* : Add 0.4 mM Methyl-b-cyclodextrin (MBCD) before use.
** : Add the essential and non essential amino acids before use.

Preparation methods of sperm preincubation and fertilization media
1. Fertilization medium: 1.25 mM GSH-mHTF medium*(1) Prepare 25 mM GSH (stock solution)
Weigh about 3 mg of GSH, and add 130 μL mHTF/ mg of GSH[Glutathione; Sigma G6013-5G), 25 mM GSH：7.683 mg/mL＝1 mg/130 μL](2) Prepare 1.25 mM GSH-mHTF medium
On the morning of experiment, add 25 μL of 25 mM GSH to 475μL of mHTF
2. Sperm preincuvation medium: 0.4mM MBCD-PVA-HTF medium**(1) Prepare 50 mM MBCD (stock solution )Add 66.7 mg of MBCD（Methyl-b-cyclodextrin; Sigma C-4555-1g）to 1 mL of PVA-mHTF(2) Prepare 0.4mM MBCD-PVA-HTF medium
On the morning of experiment, add 8 μL of 50 mM MBCD to 992 μL of PVA-mHTF
References
Hasegawa et al. J. Reprod. Dev., 58:156-161, 2012; Takeo et al. Biol. Reprod., 85:1066-1072, 2011.

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