Effects of Ascorbic Acid on Maturation Rate, Morphology, and Gene Expression of Vitrified In Vitro Matured Dromedary Camel Oocytes

Abstract

In vitro embryo generation, cryopreservation, and embryo transfer are examples of assisted reproductive technologies that can be used to improve camel genetic performance and fertility. The aim of this study was to investigate the impact of ascorbic acid supplementation to in vitro maturation media on the maturation rate, morphology, and gene expression of fresh and vitrified in vitro matured dromedary camel oocytes. In the current study, 810 oocytes of excellent and good quality were in vitro matured in maturation medium (TCM-199 + 10 ug/ml follicle stimulated hormone + 10% fetal calf serum + 100 IU/ml Pregnant mare serum + 50 μg/ml gentamycin) without any additives to act as a control group (C) and with 50 μg/ml ascorbic acid group (AA) and incubation in a CO2 incubator (38.5 ̊C, 5% CO2, 20% O2 and 95% humidity) for 40 hours. In vitro matured dromedary camel oocytes with the first polar body (n = 210) in C group and AA group (n = 250) were placed in basic medium (BM) and then placed in vitrification solution1 (VS1) for one minute, followed by the transfer of oocytes to VS2 (double concentration of VS1, containing 20% Ethyl Glycol (EG) and +20% Dimethyl sulfoxide) for 30 seconds. Oocytes were then loaded into sterile 0.25 ml straws and stored in liquid nitrogen for 7-10 days. The normal fresh and vitrified /thawed in vitro matured dromedary camel oocytes were kept in RNA later at a -80°C freezer for gene expression analysis. The maturation rate of dromedary camel oocytes in the in vitro matured AA group was significantly higher than that of the C group. The percentage of normally recovered vitrified/thawed oocytes was higher in the in vitro matured with ascorbic acid (VAA) than in the control (VC) group. The expression pattern of the SOD1 gene and GDF9 gene was upregulated in fresh AA and VAA groups than in the fresh C and VC groups. The profile of the SOD1 gene was more abundant in the vitrified/thawed oocytes VAA group than in the VC group. All vitrified/thawed groups, whether control or ascorbic acid supplemented, had lower levels of SOD1, GDF9, and BMP15 expression, compared to the fresh groups. In conclusion, the supplementation of the maturation medium with ascorbic acid has an increased maturation rate, and normal morphology of vitrified/ thawed oocytes which was linked with upregulation of SOD1, GDF9 genes expression.