Effect of cysteine,glutamate and glycine supplementation to in vitro fertilization medium during bovine early embryo development

Glutathione (GSH) is an antioxidant synthesized from three constitutive amino acids (CAA): cysteine (Cys), glycine (Gly) and glutamate (Glu). Glutathione plays an important role in oocyte maturation, fertilization and early embryo development. This study aimed to investigate the effect of Cys (0.6 mM), Gly (0.6 mM) and Glu (0.9 mM) supplementation during in vitro fertilization (IVF) of cattle oocytes. In a Pilot Experiment, de novo synthesis of GSH in bovine zygote was evaluated using a modified TALP medium prepared without MEM-essential and MEM-non-essential amino acids (mTALP): mTALP + CAA (constitutive amino acids); mTALP + CAA+5 mMBSO (buthionine sulfoximide); mTALP + Cys + Gly; mTALP + Cys + Glu and mTALP + Gly + Glu. This evidence led us to investigate the impact of CAA supplementation to TALP medium (with essential and non-essential amino acids) on zygote viability, lipid peroxidation, total intracellular GSH content (include reduced and oxidized form; GSH-GSSG), pronuclear formation in zygotes and subsequent embryo development. IVF media contained a) TALP; b) TALP + Cys + Gly + Glu (TALP + CAA); c) TALP + Cys + Gly; d) TALP + Cys + Glu; e) TALP + Gly + Glu, were used. Total GSH-GSSG concentration was increased in TALP, TALP + CAA, and TALP + Cys + Gly. The viability of zygote was similar among treatments. Lipid peroxidation was increased in zygote fertilized with TALP + Cys + Gly; TALP + Cys + Glu; TALP + Gly + Glu and TALP + CAA. The percentage of penetrated oocytes decreased in TALP + CAA and TALP + Cys + Gly. The cleavage rate was lower in TALP + CAA and TALP + Gly + Glu. The percentage of embryos developing to the blastocyst stage was lower in TALP + Cys + Glu and TALP + CAA. In conclusion, we have demonstrated the synthesis of GSH during IVF. However, Cys, Gly and Glu supplementation to TALP medium had negative effects on embryonic development.