Effect of follicle size on hormonal status of follicular fluid, oocyte ATP content, and in vitro embryo production in prepubertal sheep.

The aim of this study was to assess the effect of follicular size on estradiol (E) and progesterone (P) levels in intrafollicular fluid, ATP content in oocytes, and the embryo development rate in prepubertal sheep. Slaughterhouse ovaries were dissected to recover the follicles, which were classified according to the follicle diameter as <3 mm (n = 20) and ≥3 mm (n = 17). Then, follicular fluid was obtained and analyzed by radioimmunoassay to determine the E and P concentrations. Another group of ovaries was used to recover cumulus-oocyte complexes according to follicle size. In vitro maturation (IVM), in vitro fertilization (IVF), and embryo culture were performed using standard procedures, and ATP level was assessed at 0 and 24 h of IVM. Intrafollicular concentrations of E and P and E:P ratio were higher in ≥3 mm (18.7 ± 5.9 ng/mL, 7.8 ± 1.2 ng/mL, and 3.6 ± 1.3, respectively) than <3 mm (1.8 ± 0.4 ng/mL, 2.6 ± 0.3 ng/mL and 0.9 ± 0.3, respectively) follicles. The rate of ATP increased during IVM and was higher in oocytes from ≥3 mm than <3 mm (22.4 ± 0.7 and 8.6 ± 2.2-fold change; respectively) follicles. After IVF, the blastocyst development was higher in oocytes recovered from ≥3 mm (11.1 ± 0.9%) than from <3 mm (6.5 ± 0.7%) follicles. These results indicate an improvement in the competence and development of oocytes from ≥3 mm follicles with a higher E:P ratio. Thus, this ratio could be used as reference to design IVM medium and to enhance the in vitro embryo production in lambs.