Melatonin during bovine oocyte maturation and embryonic development improves the quality of in vitro produced embryos

Recent studies have been shown that melatonin has an important role in oocyte maturation and embryo development, mainly for its antioxidant action, however, these studies evaluated the effect of melatonin just in one stage, or in vitro oocyte maturation (IVM) or embryo culture (IVC). Therefore, the aim of this study was to evaluate the effect of melatonin on in vitro embryo production (IVP) when added in the medium of IVM and IVC. For IVP, ovaries coming from abattoir were punctured and the cumulus-oocyte complexes (COCs) were selected randomly according to Experimental Groups: 126 COCs in Control (IVM and IVC without adding melatonin); 121 COCs in Melatonin 1 (IVM and IVC with 1012M melatonin) and 121 COCs in Melatonin 2 (IVM with 1012M and IVC with 109 m of melatonin). For IVM culture medium (M199, Gibco, Carlsbad, CA, USA) was supplemented with 10% FCS, 0.5µg/ml FSH, 50 µg/ml LH, 22μg/ml pyruvate, 50μM betamercaptoethanol and 50 µg/ml gentamicin, for a period of 18 h. In vitro fertilization was used frozen semen from a single bull (Bos taurus taurus) at a concentration of 2X106 sperm/ml after separation by discontinuous Percoll density gradient. The IVF was performed in the synthetic oviductal fluid (SOF) medium according to Holm et al. (Theriogenology, 52: 683-700, 1999) without glucose and supplemented with 6 mg/mL BSA, 10µg/ml heparin, 2µM penicillamine, 1µM hipotaurina, 0,25µM epinephrine and 50 µg/ml gentamicin, for a period of 32 hours. The embryo cultures were grown in co-culture system with cumulus-oophorus-cell monolayer in SOF medium supplemented with 10% FBS, 6 mg/ml BSA, 50μM betamercaptoethanol and 50 µg/mL gentamicin. All IVP was performed in 35 mm polystyrene petri dishes, drops 100µL of culture medium under sterile mineral oil, in a culture oven with 5% CO2, 20% O2 and 75% N2, under humid atmosphere and temperature of 38.5 °C. The results of cleavage, blastocyst formation and expression of genes octamer-binding transcription factor 4 (OCT-4), interferon-tau (IFN-t) and heat shock proteins (HSP70) were analyzed with ANOVA (post test turkey, program BioEstat 5.0).The significant difference between treatments was set at P 0.05) in cleavage rates and blastocyst formation, respectively, among the Control Groups (84.92 and 46.03%), Melatonin 1 (84.30 and 40.50%) and Melatonin 2 (86.78 and 49.59%). At the molecular level, the gene expression of OCT-4 and IFN-t genes were significantly (P < 0.05) higher and lower, respectively, in Melatonin 1 compared to Control and Melatonin 2. Melatonin 2 had low HSP70 expression compared to Control and Melatonin 1. Thus, supplementation with melatonin, IVM and IVC, influence the expression of important genes that are used as indicative of embryo quality, but not altered the cleavage rate and blastocyst formation showing its importance on in vitro production of bovine embryos.