Transition of Cell Numbers in Bovine Preimplantation Embryos: In Vivo Collected and In Vitro Produced Embryos

Abstract

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 ...

The Mechanism of Oocyte Activation Influences the Cell Cycle–Related Genes Expression During Bovine Preimplantation Development

“…Ushijima et al (2008) reported differences in cell number in bovine embryos at day 8 of culture related to the method used (in vivo, IVF or NT-SC, 305.7 -29.9, 169.4 -20, and 97.8 -6.5, respectively). The fact that these genes are upregulated may suggest a failure in cell cycle control and an attempt to reestablish the normal cellular functions.…”

Classification of Morphological Changes Based on the Number of Cleavage Divisions in Bovine Embryos

“…When high-quality embryos, which are selected according to morphology, are used, the TCNs of embryos can be counted accurately [11,15,16]. Therefore, the collected embryos were classified into five quality groups (excellent, good, fair, poor and degenerated) according to the morphological evaluation method [4] using an inverted phase-contrast microscope (IMT 10; Nikon, Tokyo, Japan).…”

“…They were then mounted on a glass slide using a small amount of fixative, air-dried for 1 h at RT, and stained with Giemsa stain (Gibco-Invitrogen, Carlsbad, CA, USA) for 10 min. According to the cell-count protocol [16], TCNs of the embryos were counted under a microscope at 400× magnification. The number of CDs (CDN) calculated from the TCNs of the embryos was expressible as an equation of the first degree with a high correlation to age (y=1.03x+0.16, r=0.99) [16].…”

“…According to the cell-count protocol [16], TCNs of the embryos were counted under a microscope at 400× magnification. The number of CDs (CDN) calculated from the TCNs of the embryos was expressible as an equation of the first degree with a high correlation to age (y=1.03x+0.16, r=0.99) [16]. This result means that all bovine blastomeres divide roughly once a day for up to 9 d after ovulation.…”

“…Van Soom et al [15] suggested a strong association between CD timing and morphological changes not only in the precompaction stages but also in post-compaction stages of in vivoderived bovine embryos by showing that embryonic compaction and cavitation occur preferentially at the sixth and seventh cleavages, respectively. Recently, we demonstrated that cleavages, as calculated from the TCN of embryos, occur regularly once a day up to at least 9 days in high-quality, bovine embryos collected from superovulated donors [16]. That observation makes it possible to develop a standardized method for evaluating embryonic morphologies if a connection exists between the timing of morphological changes and the CD number.…”