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Ethanol is used routinely to dilute cell culture media supplements with little or no water solubility. This study evaluates the effect of low concentration of ethanol on the follicular development, oocyte maturation, hormone production, gene expression, and metabolomics profile of spent culture medium after long-term culture of isolated ovine preantral follicles. For this, follicles were cultured for 18 days in α-Minimum Essential Medium + alone (control treatment) or supplemented with 100 ng/mL recombinant bovine FSH (rbFSH treatment) or with 0.2%-v/v ethanol (ethanol treatment). Ethanol treatment increased the percentage of degenerated follicles and oocytes significantly, however, it showed the highest estradiol secretion. Also, the rate of meiosis resumption was higher in ethanol treatment than Control treatment. Ethanol treatment decreased the mRNA levels of B-cell lymphoma 2 (BCL2), BCL2 associated X, Aquaporin 3, Connexin 43, Inhibin Subunit Beta A, kit ligand, Heat Shock Protein (HSP A1A) significantly when compared to the Control treatment. However, mRNA levels of cytochrome P450 family 19, and FSH receptors were significantly higher in ethanol treatment than in the Control treatment. The levels of some metabolites, which are likely amino acids, lipids, an analog of Cyclic guanosine monophosphate, and a derivative of phosphoinositol phosphate metabolism, had higher relative concentrations in ethanol and rbFSH treatments than the Control treatment. In conclusion, ethanol addition augmented the follicular and oocyte degeneration rates but increased the estradiol production and the meiotic resumption. Furthermore, the follicular metabolomic profile was similar between ethanol and rbFSH treatments being both treatments; however, different from the Control treatment., (Copyright © 2020 Elsevier Ltd. All rights reserved.)