Porcine Cumulus Oocyte Complex Isolation: A Technique to Isolate Cumulus Oocyte Complex from Porcine Ovarian Follicle for In Vitro Fertilization

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Isolation of porcine cumulus-oocyte complexes

1. To collect material for the isolation of ovarian follicles, excise porcine ovaries from prepubertal gilts (approximately 6-7 months of age, weighing 70 to 80 kg) at a local slaughterhouse. Choose approximately 20 pig ovaries from 10 animals for cumulus-oocyte complexes (COCs) isolation in each experiment.
   NOTE: Assuming each ovary yields 3–5 follicles, the total number of follicles varies from 60 to 100.
2. Place ovaries in a thermos with sterile phosphate-buffered saline (PBS; pH 7.4; 38 °C) containing 1% antimycotic solution (AAS). Ensure that the experimental material is transported to the laboratory within 1 h where it is rinsed twice with sterile PBS containing antibiotics.
3. After rinsing the ovaries, transfer them to the beaker filled with handling medium (HM) (Table of Materials) and store them in an incubator at 38 °C for the time of all manipulations.
   NOTE: For optimal results during oocyte handling, carry out all procedures in DMEM/F12 medium (handling medium, HM) with the addition of 2.5% antibiotic/AAS and 10% fetal bovine serum (FBS) and control the pH at the CO₂ level in the environment, on a heating table with temperature control (38 °C) and under the laminar flow hood to minimize bacterial contamination.
4. From large porcine follicles (6-8 mm in diameter) collect follicular fluid (FF) by aspiration and centrifugation at 100 x g for 10 min at room temperature. After that, filter the supernatant using a sterile syringe attached to 0.2 μm membrane pore filters and snap freeze at -80 ° C.
   NOTE: Use an insulin syringe (u- 40) for aspiration of follicular fluid.
5. Ensure that only healthy, medium-sized follicles (4-6 mm in diameter) are selected for COCs isolation.
   NOTE: COCs of grade I, possessing an intact and round oocyte with homogeneous ooplasm and multi-layered (at least 3-4) compact cumulus are considered suitable for further 3D IVM procedure.
6. To isolate COCs, transfer 2-3 ovaries to a sterile 10 cm diameter Petri dish filled with HM. Isolate COCs from medium-sized follicles by following one of the procedures below.
   1. Gently cut the surface of the protruding ovarian follicles with a sterile surgical blade 15C. This will cause the follicular fluid along with COCs to flow out into the Petri dish.
   2. Aspirate the follicular content using 28 G needle having a size of 5/8” attached to a disposable syringe and transfer it into the Petri dish.
      NOTE: Discard the remains of ovarian tissue. Subsequent stages of isolation are carried out under a stereomicroscope.
7. Prepare 3–5 60 mm IVF Petri dishes.
   1. Add 1 mL of HM to the central wells and place 3-4 drops of HM (50 μL per drop) in their outer rings.
   2. Then, using a polycarbonate micropipette, move the undamaged COCs to drops of HM in the outer rings to rinse them briefly for 3-4 times.
   3. At the end, individually transfer them into the central well. Store these IVF plates in an incubator for further procedures.