In this study, the quality (number of cells) and pregnancy rates of bovine blastocysts produced by in vitro maturation/in vitro fertilization (IVM/IVF) following cultivation in either cell-free culture or co-culture were compared. Bovine one-cell IVM/IVF embryos obtained 6 h after insemination were stripped of cumulus cells and assigned to either cell-free culture or co-culture with granulosa cell monolayers for 9 days (Expt 1) or 10 days (Expts 2 and 3). In Expt 3, day-7 (day 0 = day of insemination) blastocysts, day-8 expanded blastocysts and day-9 hatched blastocysts were air-dried, fixed and stained to determine the number of cells. Expanded blastocysts obtained in Expt 1 were cryopreserved using propylene glycol as a cryoprotectant and were used later for embryo transfer. There were no significant differences between cell-free culture and co-culture in the percentage of one-cell embryos that developed to 2- to 16-cells (66.7% versus 72.4% for Expt 1, 71.0% versus 78.2% for Expt 2). However, significantly more (P<0.05) of the one-cell embryos co-cultured with granulosa cell monolayers developed to morula, blastocyst and expanded blastocyst stages compared with those in cell-free culture (35.0 versus 27.1%, 25.1 versus 14.2%, 15.6 versus 5.4% for Expt 1; 37.6 versus 24.0%, 25.7 versus 11.0%, 16.8 versus 3.0% for Expt 2). Only embryos co-cultured with granulosa cell monolayers hatched (Expt 2). Embryos co-cultured with granulosa cell monolayers also had higher (P<0.01) numbers of cells (92 ± 42 versus 56 ± 21 for blastocysts, 149 ± 53 versus 81 ± 29 for expanded blastocysts). Pregnancy rates tended to be higher for embryos produced by co-culture compared with those from cell-free culture (40.0 versus 27.3%, respectively). The results suggest that embryos produced in cell-free culture were of poorer quality than those produced in co-culture; however, some of them were developmentally competent as confirmed by the births of three calves after transfer to eleven recipient cows.

Summary. Bovine follicular oocytes surrounded by cumulus cells for more than one-third of their surface were matured, fertilized and developed in vitro utilizing a co-culture system with bovine cumulus cells. Embryos developed into blastocysts were non-surgically transferred to the uteri of cows at Day 6, 7 or 8 (Day 0 = oestrus). Out of 6 recipient cows (19 blastocysts transferred), 3 became pregnant. One of the 3 pregnant cows carried twins.

The results of this study demonstrated the viability of embryos obtained from in-vitro maturation of bovine oocytes followed by in-vitro fertilization and culture to the blastocyst stage in vitro.

Keywords: in-vitro; maturation; ferilization; cattle

The effect of intraoviductal embryos on endometrial receptivity was studied by intraendometrial and intrauterine embryo transfer. Five-week-old female ICR mice were mated after superovulation; a vaginal plug confirmed day 1 of pregnancy. On day 4 (90 h after hCG injection), blastocysts were collected and transferred to pseudopregnant female mice and to recipient mice in which the uterotubal junction had been ligated bilaterally on day 1 of pregnancy. Three embryos per uterine horn, a total of six embryos per recipient mouse at days 1–6, were transferred to the endometrium or uterine cavity and implantation and pregnancy rates were calculated. The implantation rate for intraendometrial embryo transfer to recipients of days 3, 5 and 6 was significantly higher for uterotubal junction-ligated mice (72.2, 20.8 and 9.7%, respectively) than for pseudopregnant mice (55.0, 8.3 and 0.0%, respectively). The implantation rate for intrauterine embryo transfer to recipients at days 2, 5 and 6 was significantly higher for uterotubal junction-ligated mice (11.1, 25.0 and 8.3%, respectively) than for pseudopregnant mice (0.0, 3.3 and 0.0%, respectively). Uterotubal junction-ligated mice achieved implantation and bore neonates by intrauterine embryo transfer on days 2 and 6, whereas no implantation was achieved in pseudopregnant mice. The difference in implantation rate could not be explained by a difference in progesterone concentration between the groups. The distribution of proliferating cells in the endometrium was also studied immunohistochemically by use of anti-proliferating cell nuclear antigen (PCNA) antibody in the recipient mice. PCNA-positive cells were more abundant in uterotubal junction-ligated mice and demonstrated a marked extension from the epithelium to the stroma over time, in contrast to those in pseudopregnant mice. These findings indicate that an intraoviductal embryo exerts a biological effect by sending a signal to the endometrial epithelium and stroma, thus facilitating endometrial receptivity to the embryo and improving the rate of implantation.