A reliable procedure for differential staining of in vitro produced bovine blastocysts: comparison of tissue culture medium 199 and Ménézo's B2 medium

A reliable double-dye technique has been established for counting the number of inner cell mass and trophectoderm cells of in vitro produced bovine blastocysts. The latter were first incubated in a 1:2 dilution of a rabbit antiserum raised against a mixture of recombinant bovine interferon tau and serum containing medium conditioned by in vitro produced trophoblastic vesicles for 45 min at 39°C. Subsequently, the blastocysts were incubated in a 5% (v/v) solution of guinea pig complement in phosphate-buffered saline containing 50 μg/ml propidium iodide for 45 min at 39°C. Then the blastocysts were transferred to ice-cold absolute ethanol containing 25 μg/ml bisbenzimide and evaluated under a fluorescence microscope. Since trophectoderm cells were permeabilised by antibody-mediated complement lysis, they were stained by propidium iodide (red or pink). Bisbenzimide can enter lysed and non-lysed cells and therefore stained also inner cell mass cells (blue) which had been protected from complement lysis by trophectoderm cells. This modified procedure proved to be very reliable for differential cell staining of bovine blastocysts produced under various culture conditions. A comparison of blastocysts produced in Ménézo's B2 vs. TCM 199 media (both supplemented with 10% serum from cows at oestrus) revealed significant (P<0.01) differences in total cell numbers (119±24 vs. 84±10; mean±SD) and in the numbers of trophectoderm cells (79±19 vs. 57±8) and inner cell mass cells (40±7 vs. 26±5) between the two culture systems. The modified staining procedure presented here is a valuable tool for evaluating the quality of in vitro produced bovine blastocysts and for improving of culture conditions.