For further analysis, to address whether the real first cleavage plane (RCP) passes through the 2PB, the embryos were kept incubating in the incubator and imaged again after their first cleavages. To determine the occurrences of the first cleavages, we used the conventional 2-D imaging every 1 h. The embryos were imaged for the second time right after the determination. For our samples, the average time interval between the first FF-OCT imaging and the occurrences of the first cleavage was 8 h. Then we quantified every RCP using the 3-D datasets obtained with the second imaging at 2-cell stage by FF-OCT. Because the two areas of nuclei of each future blastomere move apart during division in the direction that is perpendicular to the first cleavage plane, the 3-D RCP was characterized by 3-D geometry centers of two nuclei of each 2-cell stage blastomere. Also, we classified the embryos into two categories: type C, in which the RCP occurs within 30 deg of the original 2PB at the zygote stage, but not the one which has moved to another position at 2-cell stage; type D, between 30 and 90 deg. According to 3, the RCPs which belonged to type C were considered to pass through the 2PB and the RCPs which belonged to type D were considered not to pass through the 2PB. Figure 3(a) shows the schematic model and Fig. 3(b) shows the percentages of each type. 3-D reconstructions of a typical embryo of type C before and after its first cleavage are shown in Fig. 3(c) and 3(d), while Fig. 3(e) and 3(f) shows this for type D. For visualization of the two pronuclei and the nuclei of the blastomeres, a part of the embryos were removed with clipping planes. Only 27% (14 out of 51, type C) of the RCPs were formed within 30 deg of the original 2PB, whereas 73% (37 out of 51, type D: 10 between 30 deg and 60 deg; 27 between 60 deg and 90 deg) were not, as shown in Fig. 3(b). These results suggest that the RCP does not pass through the 2PB and does not strongly depend on the 2PB. On the other hand, if there still assumed to be predetermined A-V axis and the first cleavage plane is assumed to be meridional with reference to this axis, the 2PB is not a good spatial cue that marks the animal pole. At the opposite point of view, if the 2PB does mark animal pole, only a little proportion of the real first cleavage planes would align with A-V axis. Instead, the real first cleavage planes tend to occur perpendicular (between 60 deg and 90 deg) to the A-V axis more frequently.