EFFECT OF BIOPSY AND VITRIFICATION ON IN-VITRO SURVIVAL OF OVINE EMBRYOS AT DIFFERENT STAGES OF DEVELOPMENT

The objective of the present study was to assess the in vitro viabilit y of ovine embryos at different stages of development after combining cell sampling and vitrification. Precompacted morulae, compacted morul ae and blastocysts were obtained from superovulated Sarda ewes at 4, 5 or 6 d following insemination. Embryo cell biopsy was carried out in a 100-mu l drop of PBS+10% fetal calf serum (FCS) with 10 mu mol nocod azole and 7.5 mu g/ml cytochalasin-b by aspiration (3-5 cells). Embryo s were cryopreserved at room temperature after exposure of 2 solutions for 5 min, transferred into a vitrification solution, loaded into the center of 0.25-ml straws separated by air bubbles from 2 columns of s ucrose 0.5 M and plunged immediately into liquid nitrogen. In Experime nt 1, the in vitro viability of manipulated or vitrified embryos after in vitro co-culture in TCM 199 medium with 10% FCS and sheep oviducta l epithelial cells (SOEC) in 5% CO2 humidified atmosphere in air at 39 degrees C was significantly lower (P<0.05 and P<0.01, respectively) a t precompacted morula (60 and 30%) and compacted morula (62 and 39%) s tages than intact embryos at the same stages (87 and 88%). No differen ces were found at the blastocyst stage. In Experiment 2, the in vitro survival rate of precompacted morulae which were manipulated and immed iately vitrified was lower (P<0.05) than in those manipulated and, aft er a temporary period of culture, vitrified at blastocyst stage (21 vs 48%); while no differences were found at compacted morula and blastoc yst stages. The results show that 1) the stage of development influenc es the subsequent in vitro viability of manipulated and vitrified ovin e embryos, 2) temporary culture after manipulation and before vitrific ation improves the in vitro viability of embryos, and 3) the hole in t he zona pellucida resulting from biopsy does not affect blastocyst sur vival after subsequent vitrification.