Effects of changes in Ca2+ concentration of the culture medium on the in vitro development of 11-day mouse embryos

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Abstract
The present study was designed to examine the effects of changes in Ca2+ concentration in a culture medium on the development of 11-day mouse embryos (plug day = day 0). The embryos were individually cultured for 24 hours by rotating bottles using a culture medium of 25% rat serum and 75% Waymouth's medium. The Ca2+ concentration was changed by adding EGTA or CaCl2 to the Waymouth's medium. The Ca2+ concentrations examined were 0.5, 1.0, 1.5, 2.0, and 3.0 mM in 95% oxygen and 1.0, 2.0, and 3.0 mM in 60% oxygen. Development of embryos was evaluated on the basis of heartbeat, survival, growth, and differentiation. In 95% oxygen, the best survival was obtained at 1.5 mM Ca2+, while growth and differentiation were little affected by changes in Ca2+ concentrations except at 0.5 mM. In 60% oxygen, rhythmical heartbeats were difficult to maintain in many of the embryos at all Ca2+ concentrations examined, although reasonable development was obtained at 3.0 mM Ca2+. The optimal conditions were a culture medium containing 1.5 mM Ca2+ and an oxygen concentration of 95%. The addition of Waymouth's medium to the rat serum is useful in controlling the Ca2+ concentration of the culture medium.