In vitro Production of Bovine Embryos in Ascorbic Acid Containing-Culture Medium

Abstract

This study was carried out to verify the effects of addition of ascorbic acid (AA) to BARC medium used for in vitro culture of bovine embryos. Follicles (2-7 mm in diameter) from ovaries obtained from an abattoir were aspirated, and oocytes having at least 3-4 layers of unexpanded cumulus cells and homogeneous cytoplasm were washed 3 times in base maturation medium (b-MM). Groups of 15 to 25 oocytes were matured in droplets (100 ml) of b-MM with 1 mg/ml FSH (Folltropin) and 10% FCS without PVA, for 24 h. Sperm swim-up and in vitro fertilization were performed in m-DM; oocytes and spermatozoa were co-incubated for 5 h. After that, cumulus cells were removed and presumptive zygotes were cultured in droplets (50 ml) of BARC (experiment 1), supplemented with AA at concentrations of 0, 0.1, 1, and 10 mg/ml, during the step 1 (5 to 72 hours post-insemination; h pi)_. In experiment 2 and 3 the same concentrations of AA were added to BARC medium, respectively in step 2 (72 to 144 h pi) and step 3 (144 to 192 h pi). Oocytes and embryos were incubated at 5% CO₂, at 38,5^oC, under a humidified atmosphere. Embryonic development was monitored and matured oocytes that fertilized and reached morula, blastocyst and expanded blastocyst stages were recorded at 72, 144, 168, and 192 h pi, respectively, according to the defined experimental conditions. ANOVA and Bonferroni t-test were used for the statistical analysis, with p<0.05 taken as significant. In experiment 1, AA at concentration of 10 mg/ml yielded a higher (p<0.05) percentage of morulae (53.7%), blastocysts (37.2%), and expanded blastocysts (28.1%) than the other treatments, with similar results among these. No significant differences were observed among treatments in experiments 2 and 3 for any embryonic developmental stages. In conclusion, the addition of ascorbic acid to BARC culture medium increased in vitro bovine embryo production, probably due to its function of preventing or decreasing the oxidation damage.