52 THE EFFECTS OF DONOR CELL CYCLE AND THE TIMING OF OOCYTE ACTIVATION ON DEVELOPMENT OF BOVINE NUCLEAR TRANSFERRED EMBRYOS IN VIVO
K. Matsukawa A , S. Akagi B , K. Fukunari C , Y. Hosokawa D , C. Yonezawa D , S. Watanebe B and S. Takahashi BA Kochi University, Nankoku-shi, Kochi, Japan;
B National Institute of Livestock and Grassland Science, Tsukuba-shi, Ibaraki, Japan;
C Iwate Prefecture Central Livestock Hygiene Service Center, Takizawa-mura, Iwate, Japan;
D Iwate Agricultural Research Center, Takizawa-mura, Iwate, Japan
Reproduction, Fertility and Development 23(1) 132-132 https://doi.org/10.1071/RDv23n1Ab52
Published: 7 December 2010
Abstract
The cell cycle of donor cells and recipient cytoplasts are important factors affecting development of nuclear transferred (NT) embryos. We previously showed that bovine NT embryos using pre-activated cytoplasts and early G1 cells had a high in vitro developmental rate (SSR, 2008, 41st Annual Meeting). The objective of the present study was to evaluate the effects of donor cell cycle (early G1 or G0 phase) and the timing of oocyte activation on fetal development of bovine NT embryos. Adult fibroblasts from ear skin tissue of Japanese black cattle were used as donor cells. The G0 phase cells were synchronized by serum-starvation, and the G1 phase cells were prepared from actively dividing M phase cells. NT embryo production was performed by 2 kinds of protocols as follows: 1) recipient oocytes were activated by Ca ionophore (CaI), followed with cycloheximide (CH) for 2 h, and fused with synchronized donor cells followed with cytochalasin D (CD) and CH for 1 h, then CH for 4 h (pre-activated), 2) unactivated oocytes were fused with synchronized donor cells and activation was performed by CaI 1 h after fusion, followed by with CD and CH 1 h, then CH for 4 h (post-activated). After activation treatments, NT embryos were cultured in IVD101 medium for 7 days. Then, blastocysts were transferred to recipient cows. Diagnosis of pregnancy was made by ultrasonography at days 30, 60, and 90 (Day 0 = the day of embryo transfer). As shown in Table 1, the blastocyst formation rate of the NT embryos derived from early G1 cells in the pre-activated group was higher than that from G0 cells in the post-activated group (36% v. 23%, P < 0.05). After embryo transfer, 29, 67, and 50% of recipient cows were pregnant at Day 30 in G0 post-, G1 post-, and G1 pre-activated groups, respectively. However, only 1 embryo (14%) of G0 post-activated group developed to term. In conclusion, bovine NT embryos using early G1 cells and pre-activated cytoplasts showed a high blastocyst formation rate, but the full-term development of bovine NT embryos could not be improved by using early G1 cells and pre-activated cytoplasts.
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