117 EFFECT OF RESVERATROL ON THE CULTURE OF PORCINE EMBRYOS

Abstract

Resveratrol (3,4′,5-trihydroxystilbene) is a phytoalexin present in a variety of dietary sources including grapes, plums and peanuts. It was shown to exert a wide variety of pharmacological activities ranging from anti-inflammatory effects to immunomodulation and chemoprevention. The aim of this study was to investigate the effect of resveratrol on porcine embryo development. In vitro-matured pig oocytes were activated by two direct current electric pulses (60 μ seconds each) followed by an incubation with 10 μg mL^–1 cycloheximide and 7.5 μg mL^–1 cytochalasin B, for 5 h. Activated oocytes (in groups of 10) were then placed in 20 μL drops of Porcine Zygote Medium 3 (PZM-3) supplemented with various concentrations of resveratrol (0 μm, 0.5 μm, 25 μm) and cultured for 7 days. The frequency of blastocyst formation in each group was recorded and compared by Chi-square test; the total cell numbers of the blastocysts from the two best groups (control and 0.5 μm) were counted and compared by Student’s t-test. In addition, expression levels of three genes related to apoptosis (Bax, Bcl-2, and Caspase-3) were quantified in the blastocysts by quantitative real-time RT-PCR. The analysis was repeated three times, and differences in gene expression were compared by ANOVA. Differences at P < 0.05 were considered significant. Incubation of parthenotes with 25 μm resveratrol decreased blastocyst formation from 28.1% (n = 89; control group) to 4.5% (n = 66; P < 0.05). However, when parthenogenetic embryos were cultured in the presence of 0.5 μm resveratrol, blastocyst formation increased significantly: whereas only 33.0% of control embryos (n = 312) reached the blastocyst stage, this percentage in the resveratrol-treated group was 43.5% (n = 301; P < 0.05). The average total cell numbers in control and 0.5 μm resveratrol-treated blastocysts were 42.4 ± 2.1 and 44.0 ± 1.9, respectively; the difference was not statistically significant. Finally, lower expression of the Bcl-2 and Caspase-3 genes was observed in the embryos cultured with 0.5 μm resveratrol. Evidences indicated that resveratrol has a dose-dependent effect on cells. At high concentrations resveratrol exerted an antiproliferation effect, whereas at low concentration it promoted cell division. In this study a similar dose-dependent effect of resveratrol was found. The presence of 25 μm resveratrol had a negative effect on embryonic development. However, 0.5 μm resveratrol in the culture medium induced higher blastocyst formation compared with the control group. The presence of resveratrol also affected expression levels of genes related to apoptosis. Decreased levels of Bcl-2 suggest that resveratrol may suppress the mitochondria-related anti-apoptotic activity; whereas down-regulated Caspase-3 indicates a decreased apoptotic activity in the embryos treated with resveratrol. Our results suggest that the treatment of pig embryos with 0.5 μm of resveratrol has a beneficial effect on preimplantation development leading to improved blastocyst formation. The impact of resveratrol on apoptosis needs further investigation.