84 PTEROSTILBENE CAN REDUCE THE PERCENTAGE OF LIPIDS AND REACTIVE OXYGEN SPECIES IN IN VITRO-PRODUCED BOVINE EMBRYOS

Abstract

The actual challenge for the majority of research centers involves the embryo culture medium, since it is known that the culture medium plays a large role in determining embryo quality. Pterostilbene (PT) is a natural analogue of Resveratrol, an antioxidant that can reduce lipids in embryos, but no reports exist of PT being used with IVF-produced embryos or gametes. The objective of the present research was to evaluate the effect of PT in culture media CDM1 and CDM2 on embryo production, cell count, lipid accumulation, and reactive oxygen species (ROS). A total of 4 concentrations of PT and a control were evaluated, i.e. 3, 1, 0.33, 0.11, and 0 μM, in 2 separate experiments. The first experiment was performed using 6 replicates (n = 204) to evaluate blastocyst production (n = 201) and determine the percentage of lipids using the stain Sudan-Black, (n = 100). Hoechst 33258 and propidium iodide were used for determining cell counts. The second experiment was performed using 7 replicates, the effect of using PT (0.33 μM) was compared with a control with 2 O₂ concentrations (5 and 20%) for evaluating ROS production (n = 124). Blastocysts without zona pellucida were incubated 48 h at 38.5°C in PBS (without polyvinyl alcohol) with 60 μL of ro-green fluorescent protein. After incubation, 25 μL of 4′,6-diamidino-2-phenylindole (1 mg mL⁻¹) was added and incubated for 5 min. A fluorescence microscope was used and positive ROS particles digitized using Photoshop CS6 and quantified using the program ImageJ®. The data were transformed to arcsin values for subsequent analysis. In the first experiment, ANOVA and least significant difference tests were used to determine statistical significance (Statgraphics). No significant differences were found among treatments (P > 0.05) for the internal cell mass: 3, 1, 0.33, 0.11 μM PT (29.2 ± 5.2; 28.9 ± 3.8; 22.2 ± 3.2; 29.0 ± 1.7, respectively) and the control (27.0 ± 1.7). The cells of the trophectoderm did not differ (P > 0.05) between treatments (31.7 ± 4.8; 31.3 ± 3.9; 38.6 ± 3.5; 30.8 ± 1.8) and control (33.4 ± 2.1). Total cells did not differ (P > 0.05) between treatments (72.0 ± 9.8; 82.6 ± 4.3; 94.8 ± 12.8; 73.2 ± 9.2) and control (83.8 ± 7.7). Embryo production (Day 8) was greater for control (33.5 ± 3.0) versus treatments (14.1 ± 1.7; 19.4 ± 1.9; 21.1 ± 2.6; 20.8 ± 2.1) (P < 0.05); however, PT reduced the percentage of lipids (11.0 ± 0.8; 10.7 ± 0.9; 11.6 ± 1.3; 11.3 ± 1.1) within the cytoplasm of the embryos (P < 0.05) versus control (17.01 ± 1.20). In the second experiment, a factorial 2 × 2 matrix demonstrated that the O₂ concentration did not have an effect on ROS (P > 0.05); however, the PT had a significant effect on the reduction of ROS (P < 0.05), i.e. a negative correlation, r = −0.835. In summary, we determined that PT did not improve the production of blastocysts but resulted in a significant reduction of ROS and lipids.