COMPARISON OF BIOPSY METHODS TO PRODUCE I N-VITRO FERTILIZED, SEX-DETERMINED AND CRYOPRESERVED BOVINE EMBRYOS SUITABLE FOR DIRECT TRANSFER - 2ND COMMUNICATION

The objective of this study was to compare the overall efficiency and practical value of a bovine in vitro embryo production-biopsy-vitrific ation technology using two different manipulation methods for biopsy. Slaughterhouse-derived oocytes were matured in vitro, fertilized (d0) with frozen-thawed sperm and cultured on a granulosa cell monolayer. I n Experiment I, one or two blastomeres were expelled from d4 embryos b y mechanical force through a hole made by partial zona dissection (Tab le 1). Using a darning needle hole system for individual culture of bi opsied embryos from d4 to d7.5, the blastocyst per oocyte rate was 50% , and 76% of them survived subsequent vitrification and in straw direc t rehydration (Table 2). Attempts to increase the cell number of the b iopsies by further in vitro culture were unsuccessful. In Experiment I I, d7 and d8 blastocysts were manually biopsied before or after vitrif ication. When biopsy was made before vitrification, 98% of embryos sur vived manipulation and 86% of them re-expanded after vitrification and in straw dilution. Biopsy after vitrification was less efficient, as only 69% of embryos survived both processes (Table 3). The cumulative efficiency of embryo production, d7.5 biopsy and vitrification - direc t rehydration was lower (P<0.001) as that of d4 biopsy and d7.5 vitrif ication (29 vs. 38%, respectively). However, d7.5 biopsy can have more practical value as the size of the obtainable biopsy is larger and as the work-consuming long-term individual culture of the biopsied embry os may be avoided.