87 COAGULANSIN-A VIA HEAT SHOCK PROTEIN 70 INDUCTION SHOWS BENEFICIAL EFFECTS ON THE DEVELOPMENT OF BOVINE EMBRYOS IN VITRO

Abstract

Coagulansin-A (withanolide) is the steroidal lactone obtained from Withania coagulans, which belong to Solanaceae family. The coagulansin-A induces heat shock protein 70 (HSP-70), which acts as a cellular antioxidant. This study was conducted to investigate the effect of coagulansin-A on bovine oocytes maturation and embryo development in vitro. All these oocytes were aspirated from the ovaries obtained from Korean Hanwoo cows at a local abattoir. To analyse the possible beneficial effect of coagulansin-A on bovine oocytes maturation in vitro, 355 oocytes per group (control and treatment) in seven replicates were subjected with three concentrations i.e. (1, 5, and 10 µM) of coagulansin-A. The coagulansin-A was added in the in vitro-matured (IVM) media for 20 to 22 h followed by IVF for 18 to 22 h, and after fertilization the fertilized oocytes were transferred to IVC1 media for 3 days. After 3 days, the cleavage rate was checked and the 8-cell stage embryos were transferred to IVC2 media and embryo development was checked at Day 8. The culture was carried out at 5% CO₂ and 38.5°C. The results indicated that among the three concentrations of Coagulansin-A, only 5 µM remarkably (P < 0.05) improved embryo development (Day 8 blastocyst), being 27.30% and 40.01% for control and treated groups, respectively. This concentration also significantly (P < 0.05) encouraged the activation of HSP-70, having 16.44 arbitrary units (AU) and 35.41 AU integral optical density (IOD) for control and treated groups, respectively. The immunofluorescence analysis revealed that 5 µM coagulansin-A supplementation significantly (P < 0.05) inhibited oxidative stress and inflammation during bovine embryo development in vitro by decreasing IOD of 8-Oxoguanosine (8-OxoG) from 28.12 AU in control to 18.06 AU for the treated group and nuclear factor kappa B (NF-kB) IOD (P < 0.05) from 42.25 AU to 21.80 for control and treated groups, respectively. Additionally, the results obtained from terminal deoxynucleotidyl transferase TUNEL assay confirmed that coagulansin-A treatment reduced the bovine embryo DNA damage significantly (P < 0.05) from 7.4 ± 0.375 to 5.7 ± 0.287 and improved the embryo quality (P < 0.05) with mean cell numbers of 127.7 ± 4.161 and 150.1 ± 3.624 per embryo for control and coagulansin-A treated groups, respectively. This study provides new information regarding the mechanisms by which coagulansin-A promotes bovine oocyte maturation and embryo development in vitro.