3. Once fully anesthetized, remove the animal from the cage, and
place her on a heated sterile surgical field on a prone position.
Shave the dorsal skin using a clipper, and gently wipe the
shaved area with 2 Â 2 in. cotton gauzes pre-soaked into 70%
ethanol, and then apply betadine to the shaved site using a
cotton-tipped swab pre-soaked with betadine solution. Repeat
these steps two times before starting operation.
4. In order to access to the abdominal muscle, make a single
sagittal midline dorsal incision on the skin across the lumbar
area. This incision across the lumbar area will allow access to
both ovaries for orthotopic transplantation into ovarian bursa
after bilateral ovariectomy as well as simple SC or under the
renal capsule OT grafting.
5. Separate the skin from the muscle by using scissors SC from the
incision both left and right flank region of the animal. Blunt
dissect the connective tissue, and access the peritoneal cavity by
making paralumbar muscle incision (3–4 mm in mice and
5–6 mm in rats) away from the lumbar vertebra, a little below
the kidney, identify the ovary, and surrounded by fat tissue.
6. Using blunt-curved serrated forceps, gently hold the periovarian fat tissue, and pull out the ovary through the incision,
and place it on a 2 Â 2 in. cotton gauze placed on the animal,
and secure with a bulldog-clamp. The ovarian fat pad appears
little whitish than the surrounding.
7. Place the animal under the dissecting stereomicroscope using
fiber optic light source, and focus the microscope on the ovarian bursa. Apply few drops of epinephrine onto the bursal
surface with a syringe, and wait a few minutes. This would
minimize the bleeding during incision of ovarian bursa.
8. To expose the ovary, gently pinch the bursa with fine forceps,
and incise the bursa by spring-type micro-scissors adjacent to
ovarian fat pad for mouse and between the two blood vessels
for the rats due to high vascularization (see Fig. 3a). Gently
expose and excise the ovary from the ovarian bursa using a 27 G
needle tip, and discard while clamping at the ovarian hilum
with fine forceps to prevent bleeding (see Fig. 3c and Note 4).
9. After removal of the ovary from the recipient, apply few drops
of epinephrine (50 mg/mL), and wait few minutes, and touch
with hemostatic eye spears on the empty bursa to control
bleeding, and aid in blood clotting (see Note 5).
10. Locate the frozen-thawed donor ovary in the Petri dish containing TL-HEPES solution, gently place the OT by the empty
ovarian bursa using fine forceps. Gently pinch one edge of
ovarian bursa incision, and place the donor OT (orthotopically) under the bursa (see Fig. 3d). If the OT pieces do not fit
Rodent Ovarian Tissue Cryopreservation
477
place her on a heated sterile surgical field on a prone position.
Shave the dorsal skin using a clipper, and gently wipe the
shaved area with 2 Â 2 in. cotton gauzes pre-soaked into 70%
ethanol, and then apply betadine to the shaved site using a
cotton-tipped swab pre-soaked with betadine solution. Repeat
these steps two times before starting operation.
4. In order to access to the abdominal muscle, make a single
sagittal midline dorsal incision on the skin across the lumbar
area. This incision across the lumbar area will allow access to
both ovaries for orthotopic transplantation into ovarian bursa
after bilateral ovariectomy as well as simple SC or under the
renal capsule OT grafting.
5. Separate the skin from the muscle by using scissors SC from the
incision both left and right flank region of the animal. Blunt
dissect the connective tissue, and access the peritoneal cavity by
making paralumbar muscle incision (3–4 mm in mice and
5–6 mm in rats) away from the lumbar vertebra, a little below
the kidney, identify the ovary, and surrounded by fat tissue.
6. Using blunt-curved serrated forceps, gently hold the periovarian fat tissue, and pull out the ovary through the incision,
and place it on a 2 Â 2 in. cotton gauze placed on the animal,
and secure with a bulldog-clamp. The ovarian fat pad appears
little whitish than the surrounding.
7. Place the animal under the dissecting stereomicroscope using
fiber optic light source, and focus the microscope on the ovarian bursa. Apply few drops of epinephrine onto the bursal
surface with a syringe, and wait a few minutes. This would
minimize the bleeding during incision of ovarian bursa.
8. To expose the ovary, gently pinch the bursa with fine forceps,
and incise the bursa by spring-type micro-scissors adjacent to
ovarian fat pad for mouse and between the two blood vessels
for the rats due to high vascularization (see Fig. 3a). Gently
expose and excise the ovary from the ovarian bursa using a 27 G
needle tip, and discard while clamping at the ovarian hilum
with fine forceps to prevent bleeding (see Fig. 3c and Note 4).
9. After removal of the ovary from the recipient, apply few drops
of epinephrine (50 mg/mL), and wait few minutes, and touch
with hemostatic eye spears on the empty bursa to control
bleeding, and aid in blood clotting (see Note 5).
10. Locate the frozen-thawed donor ovary in the Petri dish containing TL-HEPES solution, gently place the OT by the empty
ovarian bursa using fine forceps. Gently pinch one edge of
ovarian bursa incision, and place the donor OT (orthotopically) under the bursa (see Fig. 3d). If the OT pieces do not fit
Rodent Ovarian Tissue Cryopreservation
477