ABSTRACT. This study was conducted to evaluate whether refining mineral oil and the addition of hemoglobin and/or glucose to a serumfree medium could improve in vitro-development of embryos cultured in a chemicallysemi-defined microdroplet culture system. Block strain, outbred (ICR) mouse 1-or 2-cell embryos were cultured in 5 µl droplets of Chatot, Ziomek and Bavister medium overlaid with mineral oil of different types, and preimplantation development to the blastocyst stage was subsequently monitored. In the experiment 1, either Sigma (M-8410) or BDH (GPR TM ) mineral oil with or without washing was used for embryo culture and, distilled water (DW) or culture medium was used as a washing agent. As results, better (P<0.0001) development of 1-cell embryos was found in the Sigma than in the BDH; more blastocysts developed in Sigma oil washed with culture medium than in the others (37% vs. 0%). Subsequent ly, 1-(experiment 2) or 2-cell (experiment 3) embryos were cultured in the droplets overlaid with medium-washed Sigma oil, to which 0.001 mg/ml hemoglobin and/or 5.6 mM glucose were supplemented at the 1-cell and the 4-cell stages, respectively. Regardless of embryo stages, blastocyst formation was significantly improved by the addition of hemoglobin (54 to 48% vs. 42 to 31% in 1-cell and 83 to 78% vs. 65 to 68% in 2-cell embryos) and this effect was independent of glucose addition. In conclusion, the selection and washing of mineral oil, and the addition of hemoglobin is beneficial for improving the efficacy of a drop embryo culture system using a serum-free medium. KEY WORDS: glucose, hemoglobin, mineral oil, mouse embryo, preimplantation development.J. Vet. Med. Sci. 66(1): 63-66, 2004 Technical advance in gamete manipulation is fundamental for developing novel medical biotechnologies. Lots of information has been obtained from various model studies and we have developed the mouse model system of embryo culture employing serum-free media and outbred strain (Institute of Cancer Research; ICR) [10,11]. Subsequently, a culture technique using small microdroplet of a serumfree medium has been developed. In this system, commercially-purchased mineral oil is used for preparing microdroplets, so culture medium was continuously exposes to overlaid oil throughout the culture. It is possible that the artifacts included in overaid oil directly affect on the development of embryos cultured in microdroplet by continuous exposure during in vitro-culture. This hypothesis was confirmed by recent finding showing the influence of oil on embryo development during in vitro-culture [12]. In addition, a Chatot, Ziomek and Bavister (CZB) medium [1-4] was employed as a basic medium of our culture system, but this does not contain several substrates having embryotrophic action reported in previous studies [8][9][10][11].Consequently, a randomized, prospective study was designed to further improve our embryo culture system using the microdroplet of a serum-free medium. This study was conducted to examine 1) the effects of two types ...

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