Cellular characterization of blastocysts derived from rabbit 4-, 8- and 16-cell embryos and isolated blastomeres cultured in vitro

The purpose of this study was to investigate the developmental potential of isolated rabbit blastomeres under various culture conditions to gain insig ht into their ability to form the two cell lineages of a viable blastocyst, Intact embryos at the 4-cell, 8-cell, 16-cell stages and blastomeres isola ted from 4-, 8- and 16-cell rabbit embryos (1/4, 1/8 or 1/16 blastomeres re spectively) were cultured in drops of one of three different media, each su pplemented with either fetal calf serum (FCS), bovine serum albumin (BSA) o r polyvinyl alcohol (PVA), The effects of the extracellular matrix fibronec tin (FN) on the development of isolated rabbit blastomeres were also invest igated. Supplementation of the medium with FCS yielded a higher (P < 0.05) proportion of blastocysts than BSA or PVA, predominantly from 1/4 blastomer es, No major differences were found between the three basic culture media. In 1/4, 1/8 or 1/16 blastomeres, blastocyst formation rates were greater (P < 0.05) in groups cultured in matrix-free (54.5, 59.6 and 54.6% respective ly) than in FN-coated groups (35.4, 46.0 and 26.1% respectively). Only in b lastocysts derived from 1/4 blastomeres, were the numbers of inner cell mas s (ICM) and total cells of blastocysts higher (P < 0.05) in FN-coated group s than in matrix-free groups (12.7 +/- 1.1 versus 8.5 +/- 0.7 ICM, 73.8 +/- 3.7 versus 57.8 +/- 3.3 total cells). The percentage of blastocysts derive d from single blastomeres with ICM cells decreased with increasing cell sta ge of the parent embryos in FN-coated (93.6, 78.3 and 44.0%, respectively) as well as matrix-free groups (96.2, 69.3 and 55.2%). In FN-coated groups, after 96 h (1/4) or 72 h (1/8 and 1/16) of culture, similar to 20-30% of bl astomeres did not develop into normal blastocysts but formed sheets with 30 -50 cells attached to the bottom of the dishes. These results indicate that the development of rabbit blastomeres shares important characteristics wit h those from mouse and domestic species and may thus aid in developing an e fficient culture system for blastomeres, derived from human embryos.