a. Sperm dish
Put 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 dish
Put 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 in vitro fertilization will be carried out using fresh, frozen-thawed or cold-temperature transported spermatozoa.
Please refer to the CARD MEDIUM instruction manual.
c. Washing dish
Put 4 drops (80 µL/drop) of mHTF into a dish and cover them with liquid paraffin. Place the dish in an incubator for at least 30 minutes.
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.
Note: Be sure to carry out all operations, from sacrificing the female and removing her oviducts 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 next mouse.
Use one drop of CARD MEDIUM (200 µL) per female (2 oviducts).
Note: At this stage it is important that you identify and remove any parthenogenetic oocytes. 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 to distinguish 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 the unfertilized oocyte does not have any pronuclei (C).
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): 546-51.
Takeo T., and Nakagata N. 2010. Combination medium of cryoprotective agents containing L-glutamine and methyl-β-cyclodextrin in a preincubation medium yields a high fertilization 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):1066-1072.
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.
To be used for research only. DO NOT use for human gene therapy or clinical diagnosis.