Culture of Cattle and Sheep Follicles

Preantral Follicle Isolation

  1. Transport bovine and ovine ovaries (obtained from an abattoir) in HEPES-buffered M199, at 25-30°C. In a laminar flow hood, rinse ovaries with 70% alcohol.
  2. Take slices of ovarian cortex using a scalpel and place them in dissection medium as in Protocol 1.7 (Figure 1.5).
  3. Under the dissecting microscope, isolated preantral follicles (100-200 mm) in a petri dish, from the cortical slices using fine 25-G needles attached to syringe barrels.
  4. Select follicles with an intact basement membrane and even granulosa and theca layers for culture.
  5. Culture isolated bovine preantral follicles individually in 96-well plates (Bibby Sterilin Ltd., Stone, Staffs, UK) in 250 ml of culture medium (Protocol 1.7).
  6. Incubate plates in a sterile humidified atmosphere with 5% CO2 at 37°C. Replace half of the medium every second day.

Figure 1.5. Diagram illustrating the method used to obtain individual bovine follicles.

Figure 1.5. Diagram illustrating the method used to obtain individual bovine follicles.

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