Activite, etabolique et aptitude a la congelation de l'embryon bovin produit in vitro

摘要

This paper presents the main results of a study realised to obtain a PhD thesis kept up the 23~(rd) of october at the University Pierre and Marie Curie (Paris VI). Pregnancy rates after transfer of cryopreserved of in vitro produced bovine embryo are too low to use such embryos routinely on farm. This is the main limting factor for a large commercialisation of in vitro produced embryos. The aim of this study was to determine morphology and metabolic characteristics of in vitro produced bovine embryos and to adapt freezing procedure to the specificities of their membrane properties. The mean number of nuclei of blastocysts co-cultured on Vero cells was not significantly different from in vivo produced blastocysts. Metabolic studies on glucose, fructose, and methionine, glycine didn't shown any correlation between metabolites incorporation and morphology graduation of embryos. The pregnancy rate after transfer of freshly in vitro produced embryos was 51,8 % (29/56) at 3 months. For cryopreservation studies, we defined a two-steps wise equilibration and dilution procedure. Presence of serum and sucrose may have attenuated volume cell variations during dehydratation and rehydratation processes. Pregnancy rates after transfer of one frozen-thawed embryo were 29,8 % (14/47) at day 35 and 17 % (8/47) at 3 months. Adding lecithin (with potential for lowering membrane phase transitions temperatures) to freezing medium enhanced in vitro survival rate of postthawed embryo. The beneficial effect of lecithin on in vitro survival rate was not observed on in vivo development. We obtained a pregnancy rate of 10,7 % (3/28) at day 35 and 7, 1% (2/28) at 3 months. In a second time, we tried to modify osmotics properties of cellular membraned before freezing. We hypothesized that the removal of cytoplasm lipide may enhance the tolerance of embryos to chilling. After fertilization, zygotes were partially delipidated, in vitro cultured until blastocyst stage and then frozen. After transfer, pregnancy rates were not different from non delipidated embryos. Reactive oxygen species generated during in vitro manipulation are implicated in membrane fluidity perturbations. The addition of hypotaurine identified as an antioxidant in vivo during in vitro culture enhanced the blastocyst development rate but didn't improve their cryotolerance non pregnancy rates after transfer.