SEcuRe 2.0 – A simple and economic protocol for efficient in vitro fertilization using cryopreserved mouse sperm

Prepare 20 straws for 2 sacrificed males of the same line. Use of a single male is possible as well, but the number of straws and volume of media used needs to be reduced by 50%

Mark the straws at 2.3 cm and 4.0 cm at the open end and label them at the other end (cotton plug)

Attach a 1 ml syringe to the labeled end of the straw and aspirate HTF medium until the meniscus reached the 4.0 cm mark

Aspirate a 2.3 cm air into the straw and store the assembly until required

Place a 120 μl drop of the gCPA (for preparation see Guidelines & Warnings) in a 35-mm culture dish, cover it with paraffin oil, add another 120 μl of gCPA into the drop to obtain a tall, semi-spherical 240 μl drop (for 4 cauda epididymis pooled from 2 males) and prewarm on a 37$$ °C hot plate

Sacrifice 2 males, collect the cauda epididymides and vasa deferentia in DPBS and clean them of fat and the testicular artery to avoid contaminating the semen with blood

Dry the cauda epididymides on a tissue (to avoid dilution of gCPA with DPBS), transfer to a 240 µl drop of prewarmed gCPA (on a 37 °C hot plate for at least 5 min) and make 6–7 cuts across the cauda epididymis with a pair of micro spring scissors

Place the dish on a 37 °C hot plate for 3 min and gently swirl every min for 20 sec to help the sperm disperse from the tissue

Divide the sperm suspension into 20 aliquots of 10 µl (using a pipette and 200 µl cell-saver tips) on a 10-cm culture dish lid, avoiding carryover of paraffin oil (clean the pipette tip from the outside with a tissue to remove the oil each time before placing a 10 µl aliquot on the dish lid) and any tissue (use microscope) into the aliquots

Aspirate each 10 µl drop into a separate freezing straw followed by 2.3 cm air

Seal the straws (e.g., with a heat sealer or metal balls) and place them in liquid nitrogen gas phase for 10 min. Note: We use a custom-made metal inlay for this purpose but self-made or purchased freezing canisters can be used as well (e.g., http://card.medic.kumamoto-u.ac.jp/card/english/sigen/manual/spfreeze.html#canister or KYD-S018 from Cosmo Bio)

Transfer the straws to the liquid nitrogen tank for long-term storage

Ideally, the entire IVF procedure (oocyte preincubation, sperm preincubation and fertilization) should be performed in an incubator (5% CO₂, 37 °C) at 5% O₂, but atmospheric O₂concentration have been shown to work well, too

Prepare a 35-cm culture dish (for oocytes from a maximum of 3 females) with a 90 µl drop of HTF⁺ (for preparation see Guidelines & Warnings) covered with oil (prewarmed to 37 °C) and equilibrate it for at least 20 min in an atmosphere of mixed gas (5% CO₂, 5% O₂, 37 °C)

Collect oviducts from superovulated females 15 hours after the hCG injection and clean them in DPBS

Transfer the oviducts into the paraffin oil next to the 90 μl drop of HTF⁺

Release oocyte clutches into the oil by ripping the ampulla with forceps and drag them through the oil into the fertilization drop

Incubate oocytes for 50 min before adding the sperm suspension (at least 30 min and no longer than 60 min) in an atmosphere of mixed gas (5% CO₂, 5% O₂, 37 °C)

Prepare a 35-mm culture dish (for each IVF experiment) with 90 μl c-TYH drop (for preparation see Guidelines & Warnings) covered with paraffin oil and equilibrate it overnight in an atmosphere of mixed gas (5% CO₂, 5% O₂, 37 °C)

Remove the required straw(s) from long-term storage in liquid nitrogen on the day of IVF, place in a dewar with liquid nitrogen. For thawing, remove the straw(s) from liquid nitrogen, keep it for 5 sec at room temperature and then quickly transfer it into a 37 °C water bath for 10 min

Dry the straw(s) with a tissue and cut the sealed end and the labeled end of the straw below the cotton plug

Expel 10μl sperm suspension into the center of a 90 μl c-TYH drop using a 1 ml syringe

Preincubate for 30 min in an atmosphere of mixed gas (5% CO₂, 5% O₂, 37 °C) before the IVF procedure to allow capacitation of the sperm. Note: Do not disturb the dishes containing the frozen/thawed sperm until the sperm are moving rapidly within the medium. Otherwise, the sperm will not recover full motility

Add 10 μl of the sperm suspension taken from the edge of the c-TYH drop to the oocyte clutches with the help of a 200 µl cell-saver tip and incubate for 4 hours (at least 3 hours and no longer than 5 hours) in an atmosphere of mixed gas (5% CO₂, 5% O₂, 37 °C)

Add another 10 µl of the sperm suspension to the fertilization medium if the removal of cumulus cells assessed after 20 min of incubation is poor, indicating insufficient motility or concentration of sperm

Wash embryos after the IVF procedure through 10 drops of preincubated embryo culture medium (e.g., M16 or KSOM) and incubate overnight in embryo culture medium in groups of 15–50 embryos per drop (a 30 µl drop of embryo culture medium covered with paraffin oil) in a CO₂incubator (5% CO₂, 37 °C, 95% humidity)

The day after insemination determine fertilization rates (a percentage of the total number of inseminated oocytes that developed to the 2-cell stage)

Transfer 2-cell embryos into the oviducts of pseudo-pregnant 0.5 dpc females

(The following video contains extra context and tips, as part of the protocols.io Spotlight series, featuring conversations with protocol authors.)

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