Product manual

CARD HyperOva(Superovulation Reagent for mouse)
Cat. No.

KYD-010-EX

Size:

KYD-010-EX-X51 mL
5*1 mLwww.cosmobio.com

OriginSerum of goat, Horse-derived villus gonatropin.

Composition
1.

Inhibin antiserum (Goat)

2.

Equine chorionic gonatropin.

ProtocolMaterials and Equipment:
1.

Superovulation reagent (CARD HyperOva; Cat.# KYD-010-EX)

2.

hCG (human Chorionic Gonadotropin, CG-10; Sigma) (37.5 IU/mL in sterile saline)

3.

1 mL disposable syringe

4.

FERTIUP (Preincubation medium: PM, Cat.# KYD-002-EX)

5.

CARD MEDIUM (Cat.# KYD-003-EX)

6.

mHTF (HTF (Cat. # CSR-R-B070) can be used same as mHTF.)

7.

Micropipettes

8.

Pipette tips for preparation of dishes

9.

Pipette tips for insemination (Pipette Tip Cat. # 114; Quality Scientiﬁc Plastics)

10.

Plastic dishes (35 mm X 10 mm Cat. # 430588; CORNING)

11.

Fine scissors

12.

Pair of watchmaker's #5 forceps

13.

Micro-spring scissors (5 mm blade)

14.

Dissecting needle

15.

Filter paper

16.

Glass capillaries for embryo handling

17.

Microscope

18.

Humidiﬁed incubator (37°C, 5% CO₂, 95% air)For research use only, Not for diagnostic use.
Please read this manual thoroughly before use.

Product Manual
CARD HyperOva
Cat. No. KYD-010-EX, KYD-010-EX-X5

www.cosmobio.com

Procedure:Preparation of CARD HyperOva
1.

Take CARD HyperOva® out from freezer, and thaw it in an incubator or in water bath at
37℃ for 10 minutes. Administer CARD HyperOva® to mouse within 1 hour after thawing.

2.

Before administration, invert the tube for several times in order to mix CARD HyperOva®
well.

3.

There might be some clumps in CARD HyperOva®. Avoid them when aspirating CARDHyperOva® in a sylinge (The clumps don't aﬀect the eﬀectiveness of CARD HyperOva®).

Superovulation
1.

Induce superovulation by injecting 0.1-0.2 mL of CARD HyperOva i.p. into a 26-30 days
old female mouse (counting the date of birth as day 0). (CARD HyperOva is usuallyadministered during the light cycle, between the hours of 17:00 and 18:00).

2.

Follow this up 48 hours later with a 7.5 IU i.p. injection of human chorionic gonadotropin(hCG).

Preparation of Dishes
1.

Prepare dishes as per the instructions below and keep them in an incubator (37°C, 5%
CO₂ in air) to allow them to gas-equilibrate.

2[Outside Japan] e-mail: export@cosmobio.co.jp

Phone: +81-3-5632-9617

Fax: +81-3-5632-9618

Product Manual
CARD HyperOva
Cat. No. KYD-010-EX, KYD-010-EX-X5

www.cosmobio.com

a. Sperm dishPut 1 drop (100 µL/drop) of FERTIUP (PM) into a dish and cover it with liquid paraﬃn 30 minutes
before collecting sperm, and place the dish in an incubator.

b. Fertilization dishPut 1 drop (200 µL/drop) of CARD MEDIUM into a dish and cover it with liquid paraffin 10
minutes before collecting oocytes, and place the dish in an incubator.
Note: There are three diﬀerent methods of preparing CARD MEDIUM, depending on whether invitro fertilization will be carried out using fresh, frozen-thawed or cold-temperature transportedspermatozoa.
Please refer to the CARD MEDIUM instruction manual.
http://search.cosmobio.co.jp/cosmo̲search̲p/search̲gate2/docs/KYD̲/003EX.20140808.pdf

c. Washing dishPut 4 drops (80 µL/drop) of mHTF into a dish and cover them with liquid paraﬃn. Place the dishin an incubator for at least 30 minutes.

3[Outside Japan] e-mail: export@cosmobio.co.jp

Phone: +81-3-5632-9617

Fax: +81-3-5632-9618

Product Manual
CARD HyperOva
Cat. No. KYD-010-EX, KYD-010-EX-X5

www.cosmobio.com

Collection of Spermatozoa
1.

Sacrifice 1 or 2 mature male mice (3 to 6 months old) and remove their cauda
epididymides, avoiding as much fat, blood and tissue ﬂuid as possible.

2.

Place the tissue on sterile ﬁlter paper to blot away any blood and ﬂuid.

3.

Place the removed cauda epididymides in a sperm dish containing liquid paraﬃn.

4.

Cut the duct of each cauda epididymis using a pair of micro-spring scissors, then use a
dissecting needle to gently press the surface of the cauda epididymis and release thesperm within.

5.

Use a dissecting needle to introduce the clots of spermatozoa released from the cauda
epididymides into the drop of FERTIUP (PM).

Note: The degree of fertility varies greatly depending on the spermatozoa used.
Spermatozoa with high fertility levels can be observed moving in a vortex with high motility
at the boundary of the incubation medium. Conversely, spermatozoa which display low
motility and poor homogeneity tend to have low fertility levels.
6.

Allow the sperm to capacitate by placing the suspension in an incubator (37℃ , 5% CO₂in air) for 60 minutes before insemination.

4[Outside Japan] e-mail: export@cosmobio.co.jp

Phone: +81-3-5632-9617

Fax: +81-3-5632-9618

Product Manual
CARD HyperOva
Cat. No. KYD-010-EX, KYD-010-EX-X5

www.cosmobio.com

Collection of Oocytes
1.

Sacriﬁce a superovulating mature female mouse (26-30 days old) approximately 15-17
hours after administering hCG.

2.

Dissect the mouse to expose the abdominal cavity.

3.

Move the digestive tract from inside the abdomen and expose the uteruses, oviducts
and ovaries.

4.

Remove the uteruses, oviducts and ovaries, and place them on sterile ﬁlter paper.

5.

Remove the oviducts (ampullae) only, avoiding as much fat, blood and tissue fluid aspossible.

6.

Immerse the removed oviducts in liquid paraﬃn contained within a fertilization dish.

7.

Use forceps to hold the oviduct against the base of the fertilization dish, then use adissecting needle to tear open the ampulla of the oviduct and release the cumulusoocyte-complexes (COCs) from within. Drag them into the drop of CARD MEDIUM (200µL).

5[Outside Japan] e-mail: export@cosmobio.co.jp

Phone: +81-3-5632-9617

Fax: +81-3-5632-9618

Product Manual
CARD HyperOva
Cat. No. KYD-010-EX, KYD-010-EX-X5

www.cosmobio.com

Note: Be sure to carry out all operations, from sacrificing the female and removing heroviducts to introducing the COCs into a drop of CARD MEDIUM, in the shortest time possible(within 30 seconds).
Moreover, when carrying out this process alone, do not sacrifice multiple mice at once;
instead, sacriﬁce one mouse and swiftly remove its oviducts before moving on to the nextmouse.
Use one drop of CARD MEDIUM (200 µL) per female (2 oviducts).
8.

Keep the fertilization dish including COCs in an incubator (37℃ 5% CO₂ in air) for 30-60
minutes before insemination.

Insemination
1.

Use the tip of a pipette (Pipette Tip Cat.No.114; Quality Scientific Plastics) to add
appropriate amounts (usually about 6 µL) of the sperm suspension to the drop of CARD
MEDIUM containing the COCs.

2.

Place the fertilization dish in an incubator (37°C, 5% CO₂ in air).

3.

3hours after insemination, wash the oocytes 3 times in fresh mHTF (80 µL) in a washingdish, avoiding the transfer of CARD MEDIUM.

4.

6 hours after insemination, observe the oocytes in the third drop of mHTF and remove
any parthenogenetic oocytes which have only one pronucleus.

6[Outside Japan] e-mail: export@cosmobio.co.jp

Phone: +81-3-5632-9617

Fax: +81-3-5632-9618

Product Manual
CARD HyperOva
Cat. No. KYD-010-EX, KYD-010-EX-X5

www.cosmobio.com

Note: At this stage it is important that you identify and remove any parthenogeneticoocytes. Please note that if you do not remove the parthenogenetic oocytes at this stage,
the next day they develop to the 2-cell stage, at which point it will be impossible todistinguish the fertilized oocytes from the parthenogenetic oocytes.
Note: The fertilized oocyte has both a male and female pronucleus (A).
On the other hand, the parthenogenetic oocyte has only one pronucleus (B) and theunfertilized oocyte does not have any pronuclei (C).
5.

After overnight culture of the oocytes, transfer the obtained 2-cell stage embryos only tothe fourth drop of mHTF in a washing dish. These embryos can be vitriﬁed, transferred
to recipient females, or cultured to the blastocyst stage. (Please refer to the chapters ofSimple Vitriﬁcation of Mouse Embryos and Embryo Transfer into the Oviduct.)

References•

Takeo T., Hoshii T., Kondo Y., Toyodome H., Arima H., Yamamura KI., Irie T., and Nakagata N.
2008. Methyl-beta-cyclodextrin improves fertilizing ability of C57BL/6 mouse sperm after
freezing and thawing by facilitating cholesterol eﬄux from the cells. Biol Reprod. 78(3): 54651.

•Takeo T., and Nakagata N. 2010. Combination medium of cryoprotective agents containing
L-glutamine and methyl- β -cyclodextrin in a preincubation medium yields a highfertilization rate for cryopreserved C57BL/6J mouse sperm. Lab. Anim. 44(2): 132-7.

•Takeo T., and Nakagata N. 2011. Reduced glutathione enhances fertility of frozen/thawed
C57BL/6 mouse sperm after exposure to methyl-beta-cyclodextrin. Biol Reprod. 85(5):10661072.

•Takeo T., Nakagata N. 2015 Superovulation Using the Combined Administration of Inhibin
Antiserum and Equine Chorionic Gonadotropin Increases the Number of Ovulated Oocytes
in C57BL/6 Female Mice. PLoS One. 2015 May 29;10(5):e0128330. doi: 10.1371/journal.
pone.0128330.
12077

For research use only, Not for diagnostic use.

www.cosmobio.com