The present study was conducted to describe an original perfusion system for the culture of whole bovine ovaries for up to 96 h. Ovaries were divided into two groups: control; physiological NEFA concentrations (150 µM NEFA, comprising 25 µM SA, 50 µM PA, and 75 µM OA), and elevated NEFA concentration (425 µM NEFA, comprising 75 µM SA, 150 µM PA, and 200 µM OA). To evaluate expression of energy metabolism-related genes (H2AFZ, PLIN2, MNSOD, CPT1A, ACSL1, LDHA, GPX1, ACCA, GADPH, SLC2A1, G6PD, DNMTA3), the oocytes and granulosa cells were collected from the perfused ovaries. After end of culture, in order to evaluate 17β-estradiol (E2) and progesterone (P4) secretion, and follicular morphology, medium samples and sections from ovarian tissue were collected. It was found that expression of genes related to metabolism of energy was modified in NEFA-exposed oocytes and granulosa cells. A significant increase in mRNA expression of LDHA and and DNMTA3 was observed in oocytes exposed to elevated NEFA medium (p<0.05), while the remaining of examined genes were not affected by NEFA concentrations in oocytes (p>0.05). Moreover, it was found that granulosa cells extracted from ovaries exposed to elevated NEFA medium displayed a significant increase in mRNA expression of GADPH, LDHA, SLC2A, MNSOD and G6PD, in contrast expression of CPT1A and H2AFZ were significantly higher in control group (p<0.05). The results showed an increase in cell apoptosis in elevated NEFA, as compared to control group (p<0.05). Data showed a decreased cell proliferation after incorporation of NEFA in culture medium. Morphological analysis of normal primordial and primary follicles in both control and elevated NEFA groups were similar, while a significant decrease of normal secondary, antral and total follicles was observed in elevated NEFA group, compared with those of control group (p<0.05). The current investigation indicates that an ex vivo culture of whole ovaries in bovine is feasible. Our data als
