208 TIME LAPSE CINEMATOGRAPHIC ANALYSIS OF CLEAVAGE AND BLASTULATION IN BOVINE EMBRYOS OBTAINED BY OVUM PICKUP AND IN VITRO FERTILIZATION

Abstract

Since the 1980s, several different bovine in vitro embryo production systems have been developed, and more than 291 000 embryos have been transferred throughout the world (Thibier M 2007 IETS Newsletter 25(4), 15–20). However, we have limited knowledge about the cleavage pattern of the first, second, and third cell divisions and the developmental activities of embryos during in vitro culture (IVC). The present study was conducted to determine the developmental activities of bovine embryos obtained by ovum pickup (OPU), in vitro maturation (IVM), and in vitro fertilization (IVF). We analyzed embryonic development by time-lapse cinematography (TLC). A total of 92 cumulus–oocyte complexes were collected by OPU from Japanese Black cows and were subjected to IVM and IVF as reported previously (Imai et al. 2006 J. Reprod. Dev. 52(Suppl.), S19–S29). Inseminated oocytes were cultured in microdrops of CR1aa medium supplemented with 5% calf serum covered by mineral oil in 5% CO₂ in air at 38.5°C. Kinetics of embryo development were measured by TLC for 168 h after IVF by using a Cultured Cell Monitoring System (CCM–M1.4ZS, Astec, Fukuoka, Japan). A total of 672 photographs of the embryos were taken (1 photograph every 15 min) during IVC. Image stacks were analyzed by the CCM–M1.4 software. Timing of the first, second, and third cell divisions, blastulation, and embryonic contractions were recorded. The results are reported as time (h) passed after insemination. In total, 75 (81.5%) embryos cleaved and 61 (66.3%) embryos developed to the blastocyst stage. The first, second, and third cell divisions in these viable embryos occurred at 24.0 ± 0.5, 32.1 ± 0.2, and 39.4 ± 0.4 h (mean ± SE) after IVF, respectively. On the other hand, in nonviable embryos (those that failed to develop to the blastocyst stage; n = 14), these cell divisions occurred at 29.5 ± 2.2, 41.3 ± 3.3, and 57.2 ± 7.6 h after IVF, respectively. There tended to be a difference (P = 0.06; paired t-test) in the timing of the first cell division between viable and nonviable embryos. Blastulation of embryos began at 114.4 ± 1.1 h, embryos developed to the blastocyst stage at 127.3 ± 1.4 h, and blastocysts began to expand at 138.4 ± 1.7 h after IVF, respectively. During blastocyst development, embryonic contractions (shrinkage attributable to the rupture of the blastocoele) and tight-shrinkage (shrinking of the embryo to less than 70% of its surface area) were observed in all embryos. The mean numbers of contractions and tight-shrinkages in blastocysts were 5.3 ± 2.7 and 2.1 ± 1.0 times, respectively. The frequency of contractions from the beginning of blastulation to the blastocyst stage was significantly lower (P < 0.01) than after the blastocyst stage. It took 6.9 ± 4.6 h for the embryos to re-expand after the tight-shrinkages. These results indicate that viable in vitro-produced embryos can be selected at early stages by TLC. Further studies are necessary to clarify the importance of the pulsating activity in OPU–IVF embryos.

This work was supported by the Research and Development Program for New Bio-industry Initiatives.