The objective of this study was to improve culture condition of in vitro produced bovine embryo cultured in CR1aa and to examine the developmental ability of the oocytes from a small number of oocytes per drop culture to blastocyst stage in vitro. Bovine cumulus-oocytes complexes (COCs) were matured, fertilized in a 100 μl-drop (10 presumptive COCs/drop) of medium. After insemination, zygotes were divided into three groups; (1) CR1aa: zygotes were cultured in 100 μl-drop of CR1aa (2) CR1aa+TCM: at 72 h after culture in CR1aa, embryos were cultured in TCM (3) TCM: zygotes were cultured in TCM. The blastocyst production rate was significantly higher (p<0.05, P<0.01) with CR1aa+TCM (42.4%) than those of CR1aa+CR1aa and TCM+TCM (31.3, 22.5%, respectively). To examine the effect of number of oocytes on the IVMFC, zygotes were cultured 2-9 or 10 embryos per 100 μl drop in CR1aa+TCM for 11 days. The proportion of embryos cleaved and blastocysts were not different between embryos derived from 10 embryos/drop and 2-9 embryos/drop (80.9% vs. 13.6% and 65.5% vs. 13.7%, respectively). Four blastocysts developed from 2-9 embryos per drop were transferred into recipients. One of the 4 recipients became pregnant. One male live calf was born at 286 day. These results indicate that CR1aa+TCM are effective media for IVC of a small number of embryos per drop, and embryos from a small number of embryos per drop have the developmental capacity to become calf.

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In this paper, we show the technical art of ICSI at our laboratory. We show the basic principal of ICSI on mammalian eggs, especially mouse and bovine ICSI. The use of Piezo-micromanipulator is useful for mouse and bovine ICSI, and how to use of piezo-electric actuator is shown carefully in this paper. The important point of mouse ICSI is the survival of eggs after sperm injection. On the other hand, in bovine ICSI, the accurate sperm injection and the less injection volume by using the piezo-electric actuator are important for successful bovine ICSI.

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[in Japanese]

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The cryopreservation of supernumerary human blastocysts produced in vitro has been essential in assisted reproductive technology. In this case report, we report the clinical results of the transfer of expanded blastocysts that were cryopreserved by a slow-freezing method or a vitrification technique after artificial shrinkage. The post-thaw survival and re-expansion rates in the slow-freezing group (100 and 60%) were comparable with those in the vitrification group (95 and 95%). However, although no pregnancy was achieved in the slow-freezing group, high implantation and clinical pregnancy rates (44 and 60%) were achieved in the vitrification group.

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