Abstract. Quantification based on cleavage division (CD) of bovine preimplantation embryos facilitates quantitative analyses of embryonic developmental processes because CD occurs roughly once each day for all blastomeres for up to at least 9 days after ovulation. Therefore, embryonic morphological changes during this period were classified according to CD number. In this study, embryos collected from superovulated donors 0-9 days after ovulation were first classified morphologically into 14 conventional developmental stages. The total cell numbers (TCN) of embryos were measured using the air-dry method. The respective CD numbers of the embryos were then determined using logarithmic transformation of the TCN. The CD numbers of embryos were increased 0-10th with 11 stages. The 0th CD corresponded to 1-cell stage embryos; the 1st CD corresponded to 2-cell stage embryos; the 2nd CD corresponded to 3-4-cell stage embryos; the 3rd CD corresponded to 5-8-cell stage embryos; the 4th CD corresponded to 9-16-cell stage embryos, the 5th CD corresponded to morulae (17-32-cell stage embryos); and the 6th CD corresponded to the compact morulae. Furthermore, the 7th CD included early blastocysts to blastocysts. The 8th CD included expanded, collapsed and hatching blastocysts. The 9th CD included hatched blastocysts. The 10th CD included expanding-hatched blastocysts. The relationship between the CD number and the morphological characteristics of the bovine embryos 0-9 days after ovulation was expressed using a linear equation, and this revealed a high degree of correlation (y=0.98x-0.96, r=0.99). These results suggest that morphological changes of bovine embryos can be classified accurately using an 11-stage classification system based on the number of cleavages.

Abstract. The total cell numbers (TCNs) of bovine embryos collected from superovulated donors (VIVO embryos) were counted 0-9 d after ovulation to quantify the developmental process. Using numerical analysis of embryo development, we also compared the developmental process of VIVO embryos, in vitro-fertilized (IVF) embryos and nuclear transfer (NT) embryos obtained from enucleated oocytes and blastomere nuclei. The TCNs of embryos were measured using the air-dry method. Cleavage divisions (CD) of the embryos were obtained using logarithmic transformation of the TCN. The TCN of the VIVO embryos increased significantly (P<0.001) with time. The relationship between the CD of the VIVO embryos at 0-9 d after ovulation and age in days was described by a linear equation with a high correlation (y=1.03x+0. 16, r=0.99), showing that CD occurs about once each day for all blastomeres. However, compared to the VIVO embryos, the TCN of the IVF embryos did not increase from 3-4 d nor after 7 d; the TCN of the NT embryos did not increase after 7 d (P>0.05). The results suggest a delay in development at these developmental stages. The slopes of regression lines of the IVF and NT embryos were significantly (P<0.001) smaller, indicating that quantification of the developmental process of VIVO embryos according to TCN and CD would be useful as criteria for numerical evaluation of the developmental process of bovine in vitro produced embryos. Key words: Bovine, Cleavage division, Preimplantation embryo, Total cell numbers (J. Reprod. Dev. 54: [239][240][241][242][243] 2008) VP embryos such as in vitro maturation/in vitro fertilization/in vitro culture (IVM/IVF/IVC) embryos [1, 2] and nuclear transfer (NT) embryos [3,4] have been shown to lower developmental ability [5,6], lower tolerance to low temperatures and freezing [7,8] and lower pregnancy rates [3,9] compared to in vivo-collected (VIVO) embryos. Therefore, improvements of IVP systems and methods of evaluating produced IVP embryos in the livestock industry to produce calves might be reexamined based on the development of VIVO embryos.Because the preimplantation embryo grows by self-replicating its blastomeres [10,11], total cell numbers (TCN) of embryos and cleavage divisions (CD) of embryos, as calculated from TCN, are commonly used to evaluate the developmental ability of IVC embryos [5,8,12]. A regression relationship between CD and embryo age in days also allows expression of the embryonic developmental process as segmental speed of blastomeres. Therefore, this numerical evaluation is useful for comparing the developmental ability and production methods of embryos [13][14][15]. However, bovine VIVO embryos used in TCN measurements are limited to those at a certain period in the late stage of culture [15], with no one yet having obtained measurements for all developmental stages of IVP embryos continuously during the 0-9 d period. Consequently, we consider that establishing an optimal timeline based on bovine VIVO embryos using the TCN and CD of embryos would be useful ...

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