Successful in vitro growth of rat two-cell embryos to blastocysts using a simple chemically defined medium

Abstract

The aim of the present study was to formulate a simple chemically defined medium for the in vitro growth of rat two-cell embryos to blastocysts. Embryos from day 2 pregnant rats were retrieved and placed in paraffin oil-covered droplets of "rat two-cell embryo culture medium" (R2ECM) containing combinations of various serum supplements, glucose, L-glutamine, and cultured up to 96 h in a CO2 incubator. Embryos cultured in the basic medium (R2ECM), as well as those supplemented either with fetal bovine serum (FBS) or male rat serum (MRS) did not develop beyond the two- to four-cell stage. In R2ECM with 0.3% bovine serum albumin (BSA) and 7.5 mM glucose, 44% of embryos reached the blastocyst stage by 96 h in culture, and the blastulation rate increased to about 83% when 1 mM of L-glutamine was added. To evaluate the effects of varying doses of glucose, two-cell embryos were cultured in R2ECM supplemented with 0.3% BSA, 1 mM L-glutamine, and 2.5, 5.0, or 7.5 mM of glucose. The percentage of embryos reaching the blastocyst stage for 2.5, 5.0, and 7.5 mM glucose was 64.6%, 65.3%, and 82.9%, respectively. The present study showed that the modified medium (R2ECM) is a simple chemically defined medium that is capable of supporting in vitro growth of rat two-cell embryos to blastocysts in high proportion (greater than 80%) without the need for change of medium within 96 h of culture. © 2001 Elsevier Science Inc.