Uso de etilenoglicol monometiléter como crioprotetor na vitrificação de embriões bovinos produzidos in vitro

Abstract

Vitrification protocols for in vitroproduction (IVP) of bovine embryos are limited. The objective of this thesis was to identify a novel protocols for vitrification of IVP bovine embryos. In the first study (S1), the toxicity characteristics of the Ethylene glycol monomethyl ether (EGMME) were determined after pre-exposure of embryos to solutions containing that cryoprotectant. In the first experiment of S1, hatching rates for embryos exposed to solutions including 5%, 10%and 15% EGMME for 5 min at 39 °C (30.5%, 33.9% and 26.3% respectively) were similar (P<0.05) to those for unexposed embryos (46.9%). In the second experiment of S1, hatching rates for unexposed embryos (33.4%) and for embryos exposed to 10% EGMME (32.9%)and 15% EGMME (25.1%) were greater (P <0.05) than the rate for embryos exposed to 20% ethylene glycol (EG), a commonly used cryoprotectant (17.0%).The expression of the Bcl-2 (inhibitor of apoptosis), Bax (apoptosis promoter) and CCND2 (cell proliferation marker) genes did not differ amongthe groups (P> 0.05). In the second study (S2), EGMME was tested in vitrificationprotocolos, associated with Dimethyl sulfoxide (DMSO), with evaluation of hatching rates, gene expression and implantation vitrified of embryos in previously synchronized cows. In the first experiment of S2, embryos unexposed to EGMME were compared against five treatments with exposure to solutions containing: 20%EG and 20%DMSO; 20% EGMME and 15%DMSO; 15% EGMME and 15% DMSO; 15% EGMME and 20% DMSO; and 10%EGMME and 20% DMSO. Hatching rates were similar across all those treatments (P>0.05). In the second experiment of S2, the hatching rate of non-vitrified embryos (63.8%) was greater (P<0.05) than the rates observed for vitrified embryos conditioned in solutions containing 20% EG and 20% DMSO (T2)and 20% EGMME and 20% DMSO (T3) (37.6% and 22.0%, respectively). The rates of T2 and T3 were similar (P>0.05), but were greater than the 10.3% rate observed for T4 (15% EGMME and 20% DMSO). The expression of the BAX (apoptosis promoter) and CCND2 (cell proliferation marker) genes were similar across treatments (P>0.05). but the Bcl-2 gene (inhibitor of apoptosis) had lower expression in T4 than in the other treatments (P<0.05). Thirty days after implantation of embryos from T2 and T3, pregnancy rates of previously synchronized cows were equal to 26.6% for both groups (no embryos from T4 were used). This thesis concluded thatEGMME can be used as cryoprotectant in vitrificationprotocolos for IVP of cattle embryos because no toxicity was observed at the tested concentrations.