Ultrasound-guided transvaginal follicular oocyte aspiration combined with in vitro maturation/in vitro fertilization/in vitro culture (IVM/IVF/IVC) was used to obtain bovine embryos. The effects of aspiration frequency and FSH stimulation on follicular development, oocyte recovery and embryo development following IVM/IVF/IVC were evaluated. In Experiment 1, transvaginal follicular aspiration was performed once or twice weekly with or without FSH stimulation in the same cows (2 cows). During the 1st session (5 aspirations/2.5 weeks), follicular aspiration was performed twice a week following the administration of FSH for 2 days (a total of 18 mg FSH). During the 2nd session (5 aspirations/2.5 weeks), follicular aspiration was performed twice a week without FSH treatment. During the 3rd session (5 aspirations/5 weeks), follicular aspiration was performed once a week without FSH treatment. During the 4th session (5 aspirations/5 weeks), follicular aspiration was performed once a week following the administration of FSH for 2 days. The follicular fluid containing aspirated oocytes was kept warm (36 C) in the 2nd, 3rd and 4th sessions but it was kept at air temperature (2-8 C, winter) in the 1st session. There were no significant differences in the mean number of follicles ≥ 2 mm in diameter during the 4 treatment sessions. The mean number of oocytes collected from each cow remained constant during the 4 treatment sessions except in the 1st session with one cow. In vitro development of oocytes collected from each cow showed similar rates of development to the morula/blastocyst stages among the last 3 treatment sessions. The lower development rate of oocytes after IVM/IVF/IVC in the 1st session may be due to the cooling of oocytes after aspiration from the ovarian follicles. This was confirmed in Experiment 2 showing that cooling (5 C, 2 h) of oocytes before IVM decreased significantly (P<0.05) the proportions of embryos that cleaved and developed to transferable stage after IVM/IVF/IVC.