151 EFFECT OF ASCORBIC ACID ON OXIDATIVE STRESS AND ITS THERMOPROTECTANT ROLE ON IN VITRO EMBRYONIC DEVELOPMENT OF BUFFALO (BUBALUS BUBALIS) EMBRYOS

Abstract

The most important factors that lead to stress in farm animals are oxidative and thermal stress, leading to reduced reproductive efficiency. Oxidative stress leads to an increase in proportion of reactive oxygen species, whereas heat stress affects the physiology of animals, which lowers the conception rates of dairy cattle. In vitro culture systems have been enhanced by manipulating media with various supplements such as vitamins, growth factors, and antioxidants that have overcome these problems. Ascorbic acid has been shown to play an antioxidant role in many species such as sheep, goat, and pigs. Keeping this in mind, this study was conducted to investigate the effect of supplementation of in vitro-matured (IVM) and/or in vitro-cultured (IVC) media with ascorbic acid and evaluate its antioxidant role in in vitro development of buffalo embryos. Immature oocytes were collected from visible surface follicles (2 to 8 mm in diameter) in slaughterhouse buffalo ovaries and subjected to IVM, IVF, and IVC in a humidified CO₂ incubator at 38.5°C. Ascorbic acid was supplemented to IVM [TCM-199 + 10% featl bovine serum (FBS) + 1 µg mL⁻¹ oestradiol-17β + 5 µg mL⁻¹ pFSH + 0.81 mM sodium pyruvate + 0.68 mM l-glutamine + 50 µg mL⁻¹ gentamicin sulfate] at 50 or 100 µM or IVC (mCR2aa + 0.6% BSA + 10% FBS+ 50 µg mL⁻¹ gentamicin sulfate) at 50 µM or both IVM and IVC media at 50 µM. Oocytes without ascorbic acid were treated as the control group. Cleavage and blastocyst rate was improved when 50 µM (66.67 ± 2.27; 16.67 ± 1.26%) ascorbic acid was supplemented to IVM medium but no significant difference (P < 0.05) was observed at 100 µM (54.04 ± 2.20; 6.16 ± 0.37%) as compared with the control (62.77 ± 2.71; 10.67 ± 0.24%), respectively. When 50 µM ascorbic acid was supplemented in IVM, IVC, or both media, though cleavage rate (66.67 ± 2.27; 69.09 ± 3.22; 66.67 ± 2.23%) was similar in 3 groups, a significant increase was observed in blastocyst rate (16.67 ± 1.26; 20.18 ± 0.86; 28.57 ± 0.37%) when both media were supplemented, respectively. To evaluate the thermoprotectant effect, 4 groups were taken: group 1 without and group 2 with ascorbic acid supplementation, oocytes were given heat treatment at 39.5°C initially for 12 h during IVM; group 3 without and group 4 with ascorbic acid supplementation, oocytes were given heat treatment at 40.5°C initially for 12 h during IVM. No significant difference in developmental rate was observed at elevated temperature of 39.5°C or 40.5°C as compared with the control. Relative mRNA abundance of heat stress-related genes, HSP 70.1 and HSP 70.2, was nonsignificantly higher in oocytes matured at 39.5°C or 40.5°C after supplementation with ascorbic acid as compared to control. Relative mRNA abundance of BAX decreased at 50 µM and increased at 100 µM ascorbic acid compared with control, whereas BID showed similar results between control and treatment. Regarding anti-apoptotic gene expression, significantly higher expression was observed in MCL1 for 50 µM and lower for 100 µM ascorbic acid, and a similar nonsignificant trend was observed for BCL-XL. Developmental genes GDF9 and BMP15 showed a nonsignificant increase in 50 µM, and a nonsignificant decrease in the 100 µM supplemented group as compared with the control. Oxidative stress-related genes SOD and GPX showed a nonsignificant decrease in treated groups as compared to control. From above results, it was concluded that ascorbic acid had an anti-oxidant as well as thermoprotectant role in developmental competence that increased the potential for generation of large domestic animal in vitro embryos for research and applied technologies such as cloning and transgenesis.