RESUMO
Elevated serum non-esterified fatty acid (NEFA) levels associated with a negative energy balance (NEB) may affect ovarian function and hence reproductive performance in high-yielding dairy cows. We have investigated the individual and combined effects of the three major NEFAs on bovine theca cell proliferation and steroidogenesis in vitro. Theca cells from healthy large follicles (>8 mm) obtained from slaughterhouse ovaries were cultured in serum free medium in the presence of 0, 50, 150 and 200 microM of palmitic acid (PA; C16:0); 0, 50, 150 and 250 microM of stearic acid (SA; C18:0); and/or 0, 50, 150 and 250 microM of oleic acid (OA; C18:1). Progesterone and androstenedione concentrations were measured in spent medium after 48 h of culture and cell numbers were determined spectrophotometrically per culture well. Cell viability was assessed by annexin-V FITC/propidium iodide staining. Only the treatment with 200 microM of PA inhibited cell proliferation (P<0.001) when tested individually, both of the mixtures tested (M1=100 microM of PA, 130 microM of SA and 140 microM of OA; M2=200 microM PA, 260 microM of SA and 280 microM of OA) reduced cell numbers (P<0.001). Progesterone and androstenedione production, both per well and per 10(4) cells, were not affected by any of the treatments, with the exception of M2. This mixture reduced progesterone production per well and per 10(4) cells (P<0.05). The effects observed were most likely caused by the cytotoxic action of the NEFAs, as demonstrated by the increased percentage of early apoptotic (M1) and late apoptotic/necrotic cells (M1 and M2) in the combination treatments (P<0.05). When combined, elevated physiological concentrations of PA, SA and OA can modulate theca cell proliferation and steroidogenesis in vitro by reducing theca cell viability. These NEFAs may be one of the mediators through which NEB compromises ovarian functioning and thus fertility in high-yielding dairy cows.