Abstract

The current study was conducted to investigate the possible protective effect of curcumin supplementation on buffalo granulosa cells (GCs) under in vitro culture condition. Buffalo ovaries were collected from local abattoir in physiological saline solution and transported directly to laboratory. Follicular fluid containing GCs and cumulus-oocyte-complexes were aspirated from antral follicles with diameter 2-8 mm. The collected GCs were seeded (Approximately 375,000 viable cells) in an 8-well culture plate containing tissue culture medium-199 (TCM-199) and kept at 37 °C in a humidified atmosphere of 5% CO2. The curcumin was supplemented to TCM media at levels of 1, 2.5, 5 and 10 μM for 24 and 48 h at 37 °C or kept without treatment as control group. The viability of cells was determined using the trypan blue test. Intracellular reactive oxygen species (ROS) level was assessed by measuring the fluorescent intensity of 6-carboxy-2′,7′-dichlorodihydro fluorescein diacetate (H2DCFDA). In addition, mitochondrial activity of GCs was determined. The results of the present study indicated that the viability of GCs under culture conditions was significantly decreased in groups treated with 1, 2.5, 5 and 10 µM curcumin (86.0%, 86.26%, 83.0% and 74.0%, respectively) compared to control group (93.60 %). The two groups of granulosa cells cultured with 2.5 and 5 µM curcumin recorded greater level of mitochondrial activity than the groups cultured with 1 µM and 10 µM curcumin. Moreover, there was a significant increase in ROS level in group cultured with 10 µM curcumin, compared to control and other experimental groups. The enzyme activity of catalase (CAT), superoxide dismutase (SOD), glutathione (GSH) and 1, 1-diphenyl-2-picrylhydrazyl (DPPH) was increased after treating in vitro cultured granulosa cells with 5 µM of curcumin. However, the enzymatic activity of CAT, SOD, GSH and DPPH was declined significantly 48 h post-curcumin treatment. In conclusion, supplementation of curcumin at low concentration (2.5 µM) for 24 h to in vitro cultured GCs improved intracellular metabolic activity and antioxidant protective system, whereas it could not sustain this action for 48 h. Moreover, supplementation of curcumin at high concentration and for long duration may negatively affect viability of GCs under in vitro culture condition via induction of oxidative stress.