Three-step in vitro maturation culture of bovine oocytes imitating temporal changes of estradiol-17β and progesterone concentrations in preovulatory follicular fluid

Abstract

The objective of the article is to evaluate the effect of three-step in vitro maturation (IVM) culture system imitating estradiol-17β (E2) and progesterone (P4) concentrations in preovulatory follicles on in vitro bovine embryo production. The cumulus-oocyte complexes (COCs) were collected from follicles (2 to 8ĝ€mm in diameter) of bovine ovaries obtained from a local slaughterhouse. For IVM, the COCs were cultured for 22ĝ€h in a three-step system: (1) culture in medium 199, containing 700ĝ€ngĝ€mLĝ'1 E2 and 50ĝ€ngĝ€mLĝ'1 P4, for 5ĝ€h, followed by the medium containing 150ĝ€ngĝ€mLĝ'1 E2 and 150ĝ€ngĝ€mLĝ'1 P4 for 11ĝ€h, and then the medium containing 20ĝ€ngĝ€mLĝ'1 E2 and 300ĝ€ngĝ€mLĝ'1 P4 for 6ĝ€h (EP group); (2) culture in the medium containing 700ĝ€ngĝ€mLĝ'1 E2 for 5ĝ€h, followed by the medium containing 150ĝ€ngĝ€mLĝ'1 E2 for 11ĝ€h, and then the medium containing 20ĝ€ngĝ€mLĝ'1 E2 for 6ĝ€h (E group); or (3) culture in the medium containing 50ĝ€ngĝ€mLĝ'1 P4 for 5ĝ€h, followed by the medium containing 150ĝ€ngĝ€mLĝ'1 P4 for 11ĝ€h, and then the medium containing 300ĝ€ngĝ€mLĝ'1 P4 for 6ĝ€h (P group). The COCs were cultured in the medium containing 1000ĝ€ngĝ€mLĝ'1 E2 for 22ĝ€h (control group). After IVM, the COCs were co-incubated with sperm and further cultured. At 48ĝ€h after insemination, the cleavage rate of embryos was not different among the groups. At 192ĝ€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 E2 and P4 concentrations in preovulatory follicular fluid improves the developmental potential of embryos in vitro.