Effect of supplementation with Cysteine, Glycine and Glutamate during in vitro fertilization of bovine oocytes

ANCHORDOQUY J PATRICIO; ANCHORDOQUY J MATEO; NIKOLOFF N; CARRANZA A; PASCUA AM; FARNETANO NA; FURNUS C

Foz do Iguazu

Simposio; 10th International Ruminant Reproduction Symposium; 2018

International Ruminant Reproduction Symposium Team

The constitutive amino acids involved in glutathione synthesis are glycine (Gly), glutamate (Glu) and cysteine (Cys). In a previous study, we demonstrated that the supplementation with Cys, Gly and Glu during in vitro maturation of bovine oocytes improves its subsequent developmental capacity (Furnus etal., 2008. Metabolic requirements associated with GSH synthesis during in vitro maturation of cattle oocytes. Anim Reprod Sci. 109(1-4):88-99). The aim of this study was to evaluate the effect of Cys, Gly and Glu during in vitro fertilization (FIV). Cumulus?oocyte complexes (COC) obtained from cattleovaries collected in an abattoir were matured in vitro in TCM 199 medium containing 10% FCS and hormones. The incubations were performed at 39 ºC in an atmosphere of 5% CO2 in air with saturated humidity for 24 h. Then matured COC were washed twice in HEPES-TALP, and placed into 50 μl drops of IVF under mineral oil. COC were in vitro fertilized in a) TALP medium alone (Control) orsupplemented with b) 0.6 mM Cys + 0.9 mM Glu + 0.6 mM Gly (TALP aa); c) 0.6 mM Gly + 0.9 mM Glu (TALP Gly+Glu); d) 0.6 mM Cys + 0.9 mM Glu (TALP Cys+Glu); and e) 0.6 mM Cys + 0.6 mM Gly (TALP Cys+Gly). To determine the effect of amino acids on subsequent embryo development, the presumptive zygotes were cultured for 9 days in SOFm medium. Cleavage rates were recorded 48 h after insemination. At the end of incubations, stages of embryo development were evaluated with an inverted microscope. For this purpose, 440 COC were matured in five replicates. A completely randomized block designs were used. Statistical model included the random effects of block and the fixed effect of treatment. Cleavage, blastocyst and hatching rates were analyzed by logistic regression using GENMOD procedure (SAS Institute). Percentages of cleavage (CL) and blastocyst (BL) for Control (CL: 75.88 - BL: 36.62%); TALP Gly+Glu (CL: 65.21- BL: 32.64%); TALP Cys+Glu (CL: 59.18- BL: 21.08%), TALP Cys+Gly (CL: 71.37 - BL: 35.22%) and TALPaa (CL: 57.05 - BL: 19.66%) were significantly lower in both, TALP Cys+Glu and TALPaa, compared with Control (P0.05). In conclusion, IVF medium supplementation with Cys, Gly and/or Glu did not show an improvement effect on subsequent embryo development. On the contrary, the combination of Cys + Glu decreased cleavage and blastocyst rates.