Abstract. The objective of the article is to evaluate the effect of three-step in vitro maturation (IVM) culture system imitating estradiol-17β (E 2 ) and progesterone (P 4 ) concentrations in preovulatory follicles on in vitro bovine embryo production. The cumulus-oocyte complexes (COCs) were collected from follicles (2 to 8 mm in diameter) of bovine ovaries obtained from a local slaughterhouse. For IVM, the COCs were cultured for 22 h in a three-step system: (1) culture in medium 199, containing 700 ng mL −1 E 2 and 50 ng mL −1 P 4 , for 5 h, followed by the medium containing 150 ng mL −1 E 2 and 150 ng mL −1 P 4 for 11 h, and then the medium containing 20 ng mL −1 E 2 and 300 ng mL −1 P 4 for 6 h (EP group); (2) culture in the medium containing 700 ng mL −1 E 2 for 5 h, followed by the medium containing 150 ng mL −1 E 2 for 11 h, and then the medium containing 20 ng mL −1 E 2 for 6 h (E group); or (3) culture in the medium containing 50 ng mL −1 P 4 for 5 h, followed by the medium containing 150 ng mL −1 P 4 for 11 h, and then the medium containing 300 ng mL −1 P 4 for 6 h (P group). The COCs were cultured in the medium containing 1000 ng mL −1 E 2 for 22 h (control group). After IVM, the COCs were co-incubated with sperm and further cultured. At 48 h after insemination, the cleavage rate of embryos was not different among the groups. At 192 h after insemination, the blastocyst formation rate of EP group was significantly higher than that of the other groups. The total cell number of blastocysts did not differ among the groups. In conclusion, these results demonstrate that the three-step IVM culture system of bovine oocytes imitating temporal changes of E 2 and P 4 concentrations in preovulatory follicular fluid improves the developmental potential of embryos in vitro.

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