Antioxidative Effects of Astaxanthin against Nitric Oxide‐Induced Oxidative Stress on Cell Viability and Gene Expression in Bovine Oviduct Epithelial Cell and the Developmental Competence of Bovine IVM/IVF Embryos

The aim of the present study was to elucidate the fundamental mechanism of bovine oviduct epithelial cell (BOEC) co-culture on developmental capacity of bovine in vitro oocyte maturation/in vitro fertilization (IVM/IVF) embryos. We examined the effects of astaxanthin against nitric oxide-induced oxidative stress on cell viability by MTT assay, lipid peroxidation (LPO) by using thiobarbituric acid (TBA) reaction for malondialdehyde (MDA) and the expression of antioxidant genes (CuZnSOD, MnSOD and Catalase) or apoptosis genes (Bcl-2, Caspase-3 and Bax) by RT-PCR in BOEC. We also evaluated the developmental rates of bovine IVM/IVF embryos co-cultured with BOEC pre-treated with astaxanthin (500 μM) in the presence or absence of sodium nitroprusside (SNP, 1000 μM) for 24 h. Cell viability in BOEC treated with SNP (50-2000 μM) lowered, while astaxanthin addition (50-500 μM) increased it in a dose-dependent manner. Cell viability in astaxanthin plus SNP (1000 μM) gradually recovered according to the increase in astaxanthin additions (100-500 mM). The LPO in astaxanthin group (50-500 μM) gradually decreased in a dose dependent manner and among SNP or astaxanthin plus SNP group, SNP alone and astaxanthin (50 μM) plus SNP shown a significant increase than other groups (p < 0.05). Expression of apoptosis or antioxidant genes was detected by RT-PCR. Bcl-2 and antioxidant genes were detected in astaxanthin or astaxanthin plus SNP group, and Caspase-3 and Bax genes were only found in SNP group. When bovine IVM/IVF embryos were cultured for 6-7 days under co-culture system such as BOEC treated with astaxanthin in the presence or absence of SNP, the developmental ability to blastocysts in 500 μM astaxanthin group was the highest of all groups. These results suggest that astaxanthin has a antioxidative effect on cell viability and LPO of BOEC, and development of bovine IVM/IVF embryos due to the induction of antioxidant genes and suppression of apoptosis genes.