CULTURE OF ZONA-FREE MOUSE EMBRYOS: A COMPARISON BETWEEN DIFFERENT CULTURE SYSTEMS

Abstract

The advantages of somatic cell nuclear transfer (SCNT) without micromanipulators over the traditional SCNT with micromanipulators include decreased costs of equipment, less preparative work, a minimum requirement for skilled labor and facilitation of automation of nuclear transfer. A major technical issue for the application of SCNT without micromanipulators is culture of the reconstructed zona-free embryos, which must be cultured in isolation from each other to prevent conglomeration. However, it is known that embryo development is poor when cultured singly in microdrops. Therefore, the establishment of an individual culture system that is as efficient as group culture is urgently needed. In this study, development of zona-free zygotes from Kunming mice was compared among different culture systems. Blastocyst rates obtained with agarose-embedded, wells in the drop (a system similar to the WOW system), individual followed by group, glass capillary and microdrop individual cultures were 61%, 52%, 49%, 15% and 11%, respectively. The blastocyst cell number from agarose-embedded and glass capillary culture was significantly higher than that from other culture systems. The blastocyst rate of zona-free embryos was comparable to that of the zona intact embryos when cultured with the agaroseembedded system. When cultured with the wells in the drop system, however, the blastocyst rate of zona-free embryos was significantly lower than that of the zona intact embryos. In the individual followed by group culture system, when individual embryos were grouped at the 4-cell or early 8-cell stage, 54% and 49% developed into blastocysts, whereas only 26% developed to the blastoscyst stage when grouped at the late 8-cell stage. However, conglomeration occurred in 60% of the embryos when grouped at the 4-cell stage while almost no conglomeration was observed when grouped at the early or late 8-cell stage. Embryo recovery rates were 100%, 92% and 84% after culture with the individual followed by group, wells in the drop and agarose embedded systems, respectively. In conclusion, among the culture systems tested, the agarose embedded system supported the best development of zona-free embryos with an acceptable recovery rate, and the individual followed by group system was simple and supported fairly good embryo development but zona-free embryos must be grouped at the early 8-cell stage. The study was supported by grants from the Momentous Research Project of the China Ministry of Science and Technology (No. 2006CB944003) and from the China National Natural Science Foundation (Nos. 30430530 and 30571337). (poster)