New method for culture of zona-included or zona-free embryos: The Well of the Well (WOW) system

Culture of mammalian zygotes individually and in smalt groups results in lo wer developmental rates than culture of large groups. Zona-free zygotes als o have impaired developmental potential in current culture systems. This pa per describes a new approach to resolve the problems, the Well of the Well (WOW) system. Small wells (WOWs) were formed in four-well dishes by melting the bottom with heated steel rods. The WOWs were then rinsed, the wells we re filled with medium, and the embryos were placed into the WOWs. To test t he value of the WOW system a 3 x 3 factorial experiment was performed. Bovi ne presumptive zygotes were cultured from day I to day 7 (day 0: day of ins emination) using three modules (single embryos, embryo groups of five, or s ingle zona-digested embryos) and three different culture systems (400 mu l medium, 200 mu l drops, or WOWs). An additional control group consisted of 40 to 50 embryos cultured in 400 mu l medium. The WOW system resulted in hi gher blastocyst/oocyte rates for all three modules (single: 59%; group of f ive: 61%; single zona-digested: 53%) than the culture in drops or in wells (P < 0.05 for all). The developmental rate was independent of the number of WOWs per well. The cell number of blastocysts cultured in the WOW system d id not differ from that of the controls. Apart from its theoretical value i n revealing the role of different factors influencing embryo development in vitro, the WOW system may have immediate practical consequences in certain areas of mammalian embryo production. Mol Reprod Dev 55:256-264, 2000. (C) 2000 Wiley-Liss, Inc.