1. At least 1 h before collecting the eggs, prepare eight culture dishes of media-
2X Ml6 equilibrated in 37°C incubator.
2X Ml 6 microdrop cultures equilibrated in 37°C incubator.
4X M2 left at room temperature.
2. Kill the plugged donor females as described above.
3. Lay the animals out on absorbant paper, and soak the ventral surface of the abdomen with 70% ethanol.
4. Pinch up the skin with fingers, and make a small cut in the midline (exact position is not critical) using the regular scissors. It is then possible to skin the animal by pulling the skin away from the position of the cut toward the head and tail. The abdomen should be completely exposed. The body wall (peritoneum) should then be cut away to expose the abdominal organs. By pushing the coils of gut out of the way, the uterus and attached ovary (see Fig. 1) should be clearly visible on both sides of the abdominal cavity. The coiled oviduct is located between the ovary and uterus.
5. Gripping the uterus with watchmaker's forceps as indicated in Fig. 1, lift up the reproductive tract. This facilitates trimming of the membrane (mesometrium) that joins the reproductive tract to the body wall. The fine scissors should be used to puncture the membrane and trim it away from the oviduct.
6. Maintaining the same grip with the forceps, a cut should be made between the ovary and oviduct using the fine scissors (cut A, Fig. 1). The cut should be as close as possible to the oviduct.
7 Transferring the forceps to the oviduct itself (grip firmly but carefully), make a second cut between the uterus and oviduct (cut B, Fig. 1)
8. Place the oviduct in one of the dishes of M2.
9. Dissect out the oviduct from the other side of the animal and then proceed with the rest of the female donors. All the oviducts may be collected in the same dish of M2.
10. When viewed under the dissecting microscope (10-20Xmag.), the oviducts should appear as a mass of opaque coils with a single transparent, swollen region, termed the ampulla (see Fig. 2). The ampulla is the target for egg collection since, at this stage, it contains the cumulus mass (numerous fertilized eggs surrounded by cumulus cells). Eggs may be visible through the walls of the ampulla
11. Using one pair of forceps to hold the oviduct down, tear the ampulla with a second pair of watchmaker's forceps. Take care to orientate the tissue such that the cumulus mass is visible when it escapes from the ampulla. If the mass does not spill out immediately, it may be necessary to tease the eggs out with forceps. Discard the empty oviduct and repeat the procedure with the remaining tissues. Occasionally, an oviduct will not exhibit a prominent ampulla. In these cases, the forceps can be used to tear the oviduct apart to search for eggs. To facilitate rapid transport to a fresh dish of medium, take care to collect the cumulus masses in a single location within the first dish.
12. Using an egg transfer pipet (see Note 1), collect all the eggs and transfer to a second dish of M2.
13. Mix the cumulus masses with 50 pL of hyaluronidase delivered from a thawed tube of stock hyaluronidase using a transfer pipet. Enzymatic digestion is necessary to separate the eggs from cumulus cells. A few minutes treatment is sufficient and the eggs should not be in contact with the enzyme for a longer period. Pipetting the eggs up and down will speed the separation.
14. Transfer the eggs to a third dish of M2 using the transfer pipet. Wash the eggs by repeatedly collecting and transferring to a new location in the dish. Each time the eggs are transferred, the small cumulus cells should be abandoned such that virtually none are remaining in the final wash. Repeat with a fourth dish of M2.
15. Wash the eggs twice in one of the equilibrated dishes of Ml 6.
16. Transfer the eggs to microdrop culture (approx 30 eggs/microdrop), and incubate at 37°C in 5% CO until required for microinjection.
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