Effect of different concentrations of EGFon in vitro culture of equine ovarian follicles

The aim of this study was to evaluate the effect of different concentrations of EGF (Epidermal Growth Factor) on in vitro culture of equine preantral follicles. Ovaries (n=5) obtained at a local slaughterhouse from mares at seasonal anestrus were washed in PBS and alcohol 70 and transported in PBS plus penicillin (200 IU/mL) and streptomycin (200 IU/mL). The inner portion of ovary was divided into 11 fragments of 3x3x1 mm. One fragment of each ovary was immediately fixed in Bouin (control group, D0). The remaining 10 fragments were individually cultured in 24- well culture plates containing 1 mL MEM (Gibco BRL, Rockville, MD, USA) (osmolarity 300 mOsm/L, pH 7.2) supplemented with penicillin (100 IU/mL), streptomycin (100 mg/mL), bovine serum albumin (1.25 mg/mL- Gibco BRL, Rockville, MD, USA), ITS (Insulin- 6.25 g/mL, transferrin - 6.25 g/mL, Selenium - 6.25 ng/mL), pyruvate (0.23 mM), glutamine (2 mM) and hypoxanthine (2mM). This medium was referred as MEM +. Culture was performed for 2 or 6 days with medium change every 2 days. Medium was supplemented with different concentrations of EGF (10, 50, 100 and 200 ng/mL). After culture, fragments were fixed in Bouin and processed for histology. Follicles were classified according to the stage of development (primary or developing) and morphology (normal or degenerated). A total of 825 slides containing 3,300 tissue sections were evaluated. The statistical model used was Proportion test (P u003c 0.05). After two days of culture there was a higher proportion of viable follicles at a concentration of 100 ng/mL EGF (87.5%), while MEM had 44.4%; 10 ng/mL had 22.2%; 50 ng/mL had 46,4% and 200 ng/mL had 64.9%. We observed follicular development in all tested concentrations of EGF after two days of culture. EGF at 100 ng/ml provided the best results, with all follicles in development, while MEM had 50%, 10 ng/mL had 75%, 50 ng/mL had 69.2% and 200 ng/mL had 91.7%. After six days of culture, EGF dose did not alter follicular viability. Regarding the proportion of developing follicles after six days, the best results were obtained with EGF at 10 ng/mL and 50 ng/mL with all follicles classified as developing, and 200 ng/mL with 85.7% of developing follicles. Therefore, EGF at 100 ng/mL promoted the best viability at two days of culture, while there was no difference between treatments for this endpoint after six days of culture. Considering follicular development, EGF at 100 ng/mL was the most effective for two days, whereas the doses of 10, 50 and 200 ng/mL were most effective after 6 days of culture to promote development. We conclude that there is a dynamic demand for EGF in in vitro culture of equine preantral follicles.