136 Efficient in vitro embryo production system using in vivo-matured oocytes from superstimulated Japanese black cows

Abstract

In this study, we examined whether in vivo matured oocytes collected by ovum pickup (OPU) from superstimulated Japanese black cows can improve the productivity and quality of in vitro-produced embryos. Cows in the stimulated group received an intravaginal progesterone-releasing device (Day 0), administration of 100 μg of GnRH on Day 5, a single administration of 30 Armour units of FSH on the evening of Day 8 and prostaglandin F_2α on the evening of Day 10. The progesterone device was removed on the morning of Day11, and then 100 μg of GnRH was administered on the morning of Day 12 (0 h). The OPU and IVF were conducted at 25~26 and 30 h, respectively. Cows in the control group received no treatment before OPU, and collected oocytes were subjected to in vitro maturation followed by IVF. The cortical granules distribution of oocytes at metaphase II stage, the cleavage pattern of embryos at the first cell cycle, the developmental rate, and the quality of blastocysts were compared between the stimulated and control groups. Oocytes with cortical granules distributing cortical cytoplasm were classified as normal distribution. The cleavage pattern was evaluated at 28 h after IVF as follows: embryos with blastomeres of the same size without fragmentation were classified as normal cleavage; embryos with 2 blastomeres and several small fragments, direct cleavage from the one-cell stage to 3 or 4 blastomeres, or 2 blastomeres of different size were classified as abnormal cleavage. The developmental rate to blastocyst stage was measured on Day 9 of culture. The morphological quality of blastocysts was evaluated based on the IETS manual. All data were obtained from more than 3 replicates. In vitro development and cortical granules distribution data were analysed using chi-squared test. Other data were analysed using Student’s t-test. Normal cortical granules distribution rate in the stimulated group was higher than that in the control group (90.3 v. 23.1%; P < 0.01). Although no differences in the developmental rate to blastocyst stage (51.5 v. 58.6%) was observed, the normal cleavage rate (73.4 v. 51.2%) and the transferable embryo rate (98.3 v. 88.0%) in the stimulated group were significantly higher (P < 0.01) than those in the control group. The ratio of embryos from normal cleavage among the transferable embryos in the stimulated group was also significantly higher than in the control group (82.2 v. 57.2%; P < 0.01). In addition, the freezable embryo ratio (71.7 v. 58.1%; P < 0.072) and the total production number of embryos per head (28.0 v. 15.5; P < 0.106) showed a tendency to be higher in the stimulated group than in the control group. These results suggest that high quality embryos can be efficiently produced by the use of in vivo matured oocytes collected by OPU from superstimulated Japanese black cows.