The objectives of the present study were to investigate the effect of vitrification on the expression of the key genes associated with blastocyst developmental potential (β-catenin, E-cadherin, Oct-4, Cdx2, Gata3), and whether the presence of β-mercaptoethanol (β-ME, 100 μM) in in vitro culture (IVC) media will affect the expression of these genes. Buffalo pre-implantation embryos were divided into three groups: (1) fresh non-vitrified embryos were used as control, (2) vitrified embryos cultured with β-ME (+), and (3) vitrified embryos cultured without (-) β-ME. The results showed that all genes were affected by vitrification, however, the presence of β-ME in IVC media significantly (P < 0.05) modified the expression level of β-catenin, E-cadherin and Oct-4 in vitrified blastocyst compared to those cultured without β-ME. Protein expression analysis by immunofluorescence and western blot also revealed that the expression level of β-catenin and E-cadherin was significantly higher in vitrified embryos cultured with β-ME than those cultured without β-ME, which, in turn, was lower than fresh control group. However, there was no significant difference between vitrified groups in the expression level of Cdx2 and Gata3. Furthermore, the reduced rate of apoptosis in embryos cultured with β-ME confirms its role in protecting vitrified blastocyst against stress. In summary, vitrification alters the expression of the adhesion related genes in vitrified blastocyst, which may explain, at least in part, the reason for the low pregnancy rate following transfer of such embryos into recipient animal, and the supplementation of IVC media with β-ME significantly improved the quality of vitrified blastocyst evidenced by the modulation of the expression of blastocyst important genes, β-catenin, E-cadherin and Oct-4, and the ability to protect vitrified blastocyst against apoptosis.
Keywords: Apoptosis; Buffalo; Embryo; Implantation; β-mercaptoethanol.
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