Bisected bovine embryos were co-transferred with trophoblastic vesicles (TVs). These TVs were prepared by dissection of conceptuses that were collected by uterine flushing after culture for seven days in the uterus following transfer of embryos derived by in vitro fertilization (IVF). Pregnancy diagnoses were performed twice, between Day 26 and Day 43 and between Day 38 and Day 73 post-estrus by ultrasonography. The pregnancy rate was significantly increased at first pregnancy diagnosis when demi-embryos were transferred with TVs (66.7%, 16/24) compared with the control group (34.5%, 10/29) (P < 0.05). Three losses occurred in the co-transfer group between the first and second pregnancy diagnosis. The final pregnancy rates according to delivered offspring were 41.7% (10/24) and 27.6% (8/29), respectively. There were no statistically significant differences between the pregnant and non-pregnant groups with regard to the average diameter of the TVs measured before transfer at three points during the gestation period. The birth weight and gestation lengths of the offspring were almost the same for the co-transfer and control groups. In the co-transfer group, the genetic identities of calves from the separated embryos were not affected by the TVs, as confirmed by parental blood type testing. Delivered offspring in co-transferred groups showed normal morphology. In conclusion, the present study indicates that co-transfer of TVs prepared from conceptuses cultured in vivo following transfer of IVF embryos enhances the fertility of demi-embryos during the early stages of pregnancy, as has similarly been shown in previous research for those prepared from in vivo embryos.