| Popis: |
The aim of this study was to verify the beneficial effects of different concentrations of epidermal growth factor (EGF) on survival and antrum formation of isolated ovine preantral follicles cultured in vitro. Ovine ovaries (n = 50) were collected from a local slaughterhouse and secondary follicles (150–200 μm in diameter), without antral cavities, were mechanically isolated by microdissection using 26-gauge needles. After selection, the follicles were individually cultured in 100-μL droplets of culture medium at 39°C and 5% CO2 in air for 18 days. The basic control medium consisted of α-minimal essential medium (α-MEM) supplemented with BSA; insulin, transferring and selenium; glutamine; hypoxanthine; and ascorbic acid and then referred to as α-MEM+. For the experimental conditions, follicles were cultured in α-MEM+ alone (control) or in different concentrations of EGF (1, 10, or 50 ng mL–1). Every other day, 60 μL of the culture media was replaced with fresh media. The morphological aspects of all ovine follicles were assessed every 6 days using a precalibrated ocular micrometer in a stereomicroscope at 100× magnification. Only those follicles showing an intact basement membrane, with bright and homogeneous granulosa cells and an absence of morphological signs of degeneration, were classified as morphologically normal follicles. The rupture of the basement membrane was also observed and characterised as follicle extrusion. In addition, antral cavity formation was defined as the emergence of a visible translucent cavity within the granulosa cell layers. Data from morphologically normal follicles, extruded follicles, and antrum formation rate during in vitro culture were expressed as percentages and compared by the chi-squared test, and differences were considered significant when P |
