A RELIABLE PROCEDURE FOR DIFFERENTIAL STAINING OF IN-VITRO PRODUCED BOVINE BLASTOCYSTS - COMPARISON OF TISSUE-CULTURE MEDIUM-199 AND MENEZOB2 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 tall and serum containing medium conditioned by in vitro pr oduced trophoblastic vesicles for 45 min at 39 degrees C. Subsequently , the blastocysts were incubated in a 5% (v/v) solution of guinea pig complement in phosphate-buffered saline containing 50 mu g/ml propidiu m iodide for 45 min at 39 degrees C. Then the blastocysts were transfe rred to ice-cold absolute ethanol containing 25 mu g/ml bisbenzimide a nd evaluated under a fluorescence microscope. Since trophectoderm cell s were permeabilised by antibody-mediated complement lysis, they were stained by propidium iodide (red or pink). Bisbenzimide tan enter lyse d and non-lysed cells and therefore stained also inner cell mass cells (blue) which had been protected from complement lysis by trophectoder m cells. This modified procedure proved to be very reliable for differ ential cell staining of bovine blastocysts produced under various cult ure conditions. A comparison of blastocysts produced in Menezo'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 (1 19 +/- 24 vs. 84 +/- 10; mean +/- SD) and in the numbers of trophectod erm 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 duality of in vitro produced bovine blastocysts and for improving of culture conditions. (C) 1998 Elsevier Science B.V.