ENERGY SUBSTRATE REQUIREMENTS FOR IN-VITRO DEVELOPMENT OF EARLY CLEAVAGE-STAGE BOVINE EMBRYOS

Energy substrate preferences of bovine cleavage-stage embryos produced by in vitro maturation and in vitro fertilization were examined in a chemically-defined (protein-free) culture medium modified hamster embr yo culture medium-3, (mHECM3). Few inseminated ova cleaved without ene rgy substrates. Glucose and/or glutamine could not support embryo deve lopment, but lactate alone was effective (37% 5-8-cells), equivalent t o complex medium TCM-199 (44%). Addition of 11 selected amino acids to lactate increased embryo cleavages, although this treatment was not s ignificantly different from pyruvate alone. Addition of glucose to lac tate or to pyruvate depressed development. Lactate + amino acids was s ignificantly better than TCM-199 (54% and 26% greater than or equal to 8-cells, respectively). Blastocyst development was evaluated after tr ansferring greater than or equal to 8-cell embryos into a complex medi um (TCM-199) containing serum. Cleavage-stage embryos produced with py ruvate alone or with lactate + amino acids yielded the highest proport ions of blastocysts (36% and 41%, respectively, of inseminated ova). B etween 33-63% of blastocysts derived from embryos that were initially developed in mHECM-3 supplemented with various substrates escaped from their zonae (hatched) depending on the treatment, but none of the emb ryos from the pyruvate + glucose combination hatched. This study shows that optimal energy substrates for bovine cleavage-stage embryo devel opment can be determined using a chemically-defined culture medium, th at a simple medium with selected substrates can support early developm ent as well as or better than a complex medium, that a two-step cultur e system can be used to evaluate blastocyst development from these cle avage-stage embryos, and that timing and hatching of embryos may provi de additional information about discriminating between the suitabiliti es of different substrates for early embryo development. (C) 1996 Wile y-Liss, Inc.