Promotion of Development of Bovine Embryos Produced In Vitro by Addition of Cysteine and β-Mercaptoethanol to a Chemically Defined Culture System

Abstract

The aim of this research was to determine the effects of L-cysteine and beta-mercaptoethanol on the in vitro development of bovine embryos that had been produced in vitro. A 2 x 3 factorial arrangement of treatments was used to evaluate the effect of 0.63 or 6.9 microM L-cysteine and 0, 10, or 100 microM beta-mercaptoethanol on the development of bovine embryos in a chemically defined medium. Embryos containing 6 to 8 cells were randomly allocated to one of the six treatment combinations and were cultured for 7 d. Both beta-mercaptoethanol and L-cysteine increased the number of embryos that reached the blastocyst stage of development, although no interaction was observed between the compounds. Embryos that were cultured in the presence of beta-mercaptoethanol had more cells at the blastocyst stage than did embryos cultured in medium without beta-mercaptoethanol. These findings provide evidence that beta-mercaptoethanol and L-cysteine promote increased embryonic development and that beta-mercaptoethanol increases the number of cells in bovine embryos produced in vitro and cultured in a cell-free, protein-free culture system.

Antioxidant supplementation of culture medium during embryo development and/or after vitrification-warming; which is the most important?

“…Following cryopreservation, oocytes and embryos become especially more sensitive to the oxidative stress, resulting in lipid peroxidation, membrane injury and structural destruction [3,23,24]. Low molecular-weight thiol compounds, such as βME, maintain the redox state of cells and protect them against the harmful effects of oxidative injuries and a number of studies have indicated the promoting effects of βME during in vitro embryo production [14][15][16][17][18]. However, data on the effect of βME are conflicting or inconsistent regarding favorable concentration of βME used in culture medium, and regarding the stage of IVP in which βME supplementation better supports embryo development.…”

“…For example, Feugang et al [23] demonstrated no protective effect of βME on embryo development when added at 50 µM. In contrast, Geshi et al [16] showed that 10 µM βME in a co-culture system improves embryo development and Cammano et al [15] did not observed any difference between a low (10 µM) or high (100 µM) concentration of βME in development of resulting embryos. In addition, Geshi et al [16], Caamano et al [14] and Feuagang et al [23] reported better promoting effect of βME when added at 4-8 cell, 8-16 cell and beyond the morula stages, respectively.…”

“…Therefore, it seems that the apparent differences among studies could be attributed to the culture conditions or developmental stage in which βME has been added. In this study, 100 µM βME was chosen because it was demonstrated to be the best concentration to promote preimplantation embryo development [15,24]. Furthermore, due to increased inherent structural fragility of embryos after cryopreservation, the highest recommended concentration of βME (100 µM) was used.…”

“…Literatures reveal that although βME can directly interacts with some oxidized radicals and can chelate metallic ions, its main action is to protect oxidation of cysteine, a precursor of GSH, into cystine and increase its entry into the cell, which is known to trigger GSH synthesis [12,14,15]. GSH is a tripeptide (gamma-glutamylcysteinylglycine) and acts as a major antioxidant in the elimination of ROS and maintaining the intracellular redox state [25].…”

“…To overcome this, exogenous antioxidants such as β-mercaptoethanol (βME), a low molecular weight thiol compound, have been frequently used to increase antioxidant capacity of embryos via increasing intracellular levels of reactive oxygen species (ROS) scavengers such as glutathione (GSH) [14][15][16][17][18]. Following cryopreservation, embryos become more amenable to the deleterious effects of ROS [2,4], and as a consequence, the importance of ROS detoxification seems to be greater than the normal sate of in vitro embryo development.…”