Primary Reproductive Tissue Aquisition for Mouse In Vitro Fertilization 1
The first step in advanced mouse techno-breeding is to acquire mouse sperm.
In the animal facility you need to make embryos to give them mutations to cure disease or understand life in all its intricacies. The first step is to get some mouse sperm. This is not something most people ever even ponder. Sperm donor mice have to be euthanized. Their testicles are then removed or actually the epididymis is removed from the testicles and the sperm comes pouring out.
The epididymis is a sort of holding area where mature sperm waits to be ejaculated. It is right outside the testes. The epididymis is present in all male reptiles, birds, and mammals with normative testicular conformation. The protocol calls for mature male mice (at least 10wks old) that have not been mated for, at least, 3 days. This means that the mice of sperm gathering are forced to be celibate for three days before they were euthanized and dissected for live sperm post mortem.
Collection of Oocytes from Hyper-Ovulated Donor Females
Protocol: Mouse Ovum Collection
The hyper-ovulation or super ovulation of the donor females is achieved by giving a hormone injection to the mice the night before collection. The mice are also mated with castrated but experienced male mice (that is experienced breeder males, sterilized, without testicles or vasectomized). The night before sacrifice, spermless mating with gonadless eunuchs helps increase the egg yield as the sex act, even if a non-reproductive act, stimulates more ovulation than the use of hormones alone. At least the female egg donors have a chance at one last pleasurable night of sex before the execution. They are checked in the morning for plugs.
Dissect the oviducts from three superovulated female mice and immerse the removed oviducts in the WARM fertilization dish into a pool of liquid wax. Use forceps to hold the oviduct against the base of the fertilization dish, then use a dissecting needle to tear open the ampulla of the oviduct and release the cumulus-oocyte-complexes (COCs) from within. Drag them into WARM fertilization dish. Incubate the ova or eggs in 37C (98.6F) skim milk and sugars media. Again, it is mouse eggs in cow milk… a strange concoction.
Note: Be sure to carry out all operations, from sacrificing the female and removing her oviducts to introducing the COCs into WARM fertilization dish 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. I would like to go to Japan to see this done so quickly. Wow.
BTW, when you cut the duct of each testicle’s cauda epididymis pouch this wild freeing of highly coiled sperm tubes come undone like a crazy spring operated can of worms. When you pop the pouch of mature sperm, its like spagetti under pressure. This tightly coiled tubes filled with special storage stuff (in this case sperm) is a strong symbolic and anatomically conserved motif at all different scales: submolecular structures, anatomy, engineering and a part of the panspermia of the cosmos, compactly coild and yet often fluffy, cloudy, puff daddy on the inside.The tubes filled with sperm just keep coming out longer and longer.
Examples of Coiling in Nature and Culture:
Non-intuitive learning: the sperm is in a compacted cloud, hidden in the coiled spaghetti tubes of the epididymis.
The tubes are in the epididymis, not in the center of the testes. Sperm is not where you think it is!
Mouse sperm has a sort of hooked or barbed head which looks more like a mouse’s head/nose shape wheareas human sperm is less pointed at the tip and our faces have less pronounced head/nose shape. Coincidence or macro-micro form morphology conserved at different scales in context of develomental biological paterning? You decide.
Celibacy for three days is required before sperm acquisition. This is to get a good amount. I asked how the mice were kept from masterbating? Mice do’t masterbate with their own hands. Although they do have opposable thumbs and the agility to reach (even with whose shorty arms), they prefer frottage. In fact, a male’s sperm count will be spent if they are housed with any other mouse. They are not just interested in reproductive sex. They hump any mouse, anywhere, all the time. Stop frottage and you have a celibate mouse with excess sperm buildup and a more fertile techno-mating.
Study Question: Morphological novelties produce major new forms through variation, but if it worked at one scale it may be a structural template for another scale. Are sperm head and mature animal head related at the level of sculptural context?
Word of the Day:
Frottage: a. The act of rubbing against or touching the body of an unsuspecting person, as in a crowd, to attain sexual gratification.b. The act of consensual rubbing between two or more people, either clothed or unclothed, to attain sexual gratification.
Abbreviated In vitro Fertilization protocol –
Fresh Mouse Sperm Protocol according to Naomi Nakagata’s laboratory using freshly collected mouse spermatozoa.
Preparation of dishes
a. Sperm dish
Put 1 drop of FERTIUP into a dish warm it up in an incubator.
b. Fertilization dish
Warm it up 10 minutes before collecting oocytes.
c. Washing/incubation dish
Add 4 drops of egg rinser and warm up the dish.
Collection of Spermatozoa
Sacrifice 1 or 2 mature male mice (at least 10wks old) that have not been mated for, at least, 3 days. Keep the male alone to block frottage, enforce celibacy and deny orgasm. This allows for a ripening, a build up of sperm. Waste not, want not.
Cut the duct of each epididymis using a pair of micro-spring scissors, then use a dissecting needle to gently press the surface of the cauda epididymis and release the sperm within.
Use a dissecting needle to introduce the clots of spermatozoa released from the cauda epididymides into the warm FERTIUP in the sperm dish.
FERTIUP is warm skim milk and sugar. The fact that the mouse sperm is incubated in warm cow milk should not be passed over without some humor.
Collection of Oocytes
Dissect the oviducts from three superovulated female mice and immerse the removed oviducts in THE WARM fertilization dish into a pool of liquid wax.
Use forceps to hold the oviduct against the base of the fertilization dish, then use a dissecting needle to tear open the ampulla of the oviduct and release the cumulus-oocyte-complexes (COCs) from within. Drag them into WARM fertilization dish
Note: Be sure to carry out all operations, from sacrificing the female and removing her oviducts to introducing the COCs into WARM fertilization dish 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.
Insemination/Fertilization
This is a very abbreviated and simplified version of the Naomi Nakagata Lab fresh sperm mouse IVF protocol just to check it out. Here is the full protocol:
In Vitro means In Glass so IVF is In Vitro Fertilization or Fertilizing in Glass
Insemination
- Wait 30 minutes and add the sperm suspension to the oocytes in vitro.
- Incubate 4hrs to allow fertilization to occur.
- Wash and culture fertilized oocytes
- remove the cell debris, degenerating oocytes and dead sperm.
- Place up to 30 embryos per culture drop.
- Move the presumptive zygotes into drop 4 and culture them overnight.
Scoring the IVF:
Decide which ones look viable, healthy, worth fertilizing and in the correct phase for experimentation. Basically, grade the worthy and bleach the rest.
- Wash the worthy embryos
- transfer the high scoring embryos
- culture the fertile embryos
- or cryopreserve the viable embryos.
Add DMSO as you begin cooling to prep the eggs for Cryopreservation. DMSO is the univarsal solvent. It replaces water and allows for the cells to freeze without forming self destructing, sharp, ice crystals.
Wait 30 minutes and add the sperm suspension to the oocytes.
Incubate 4hrs to allow fertilization to occur.
Washing and culturing fertilized oocytes
remove the cell debris, degenerating oocytes and dead sperm. Place up to 30 embryos per culture drop. Move the presumptive zygotes into drop 4 and culture them overnight.
Scoring the IVF
Wash the embryos
transfer the embryos, culture the embryos or cryopreserve the embryos.
This is a very abbreviated and simplified version of the Naomi Nakagata Lab fresh sperm mouse IVF protocol just to check it out. Here is the full protocol:
EndPostNote : ICSI or Intracellular Sperm Injection has single sperm technology
Intracellular Sperm Injection is a process of single sperm isolation. Sperm incubation occurs normally but fertilisation is not simply a mixing a crowded drop of sperm with some fertiliztion ready eggs. Instead, a single sperm is captured with thickening media to immobilize it. Then a microinjector needle sucks up sperm head and tail is left sticking out. The tail is knocked or vibrated off with a needle piezo. This micro-mecha tail removal leaves you with an immobilized sperm head. This single sperm head is then injected directly into the cellular nucleus of the ovum. With the tail, a single sperm is much less manageable.
But symbolically, the process is much deeper. Who was the first single sperm catcher and who was the first single sperm tail clipper? How and why did this process develop? Can I try ICSI myself? Can I collect 5 sperm tails? How different is this from a witches recipe for the eye of a newt? In both magic and science there is an unwritten bond: the more obscure the animal, the more ornate the part, the more spectacular potency that part adds to the potion. These rare procedures draw artists and explorers of scientific culture not for what they claim to do but for the experience of a forgotten obscurity… the exhilarating process of securing the sacred aesthetic, the nuanced object of desire. Both the tail-less heads and the headless tails… this is a radically refined farming of abjection and you have to try it to really appreciate it.
