Effect Of Ascorbic Acid On Pre In -Vitro Maturation And Parthenogenetic Development Of Bovine Oocyte Derived From Small Antral Follicle

Abstract

Fully grown oocytes, derived from large follicles (4-6mm), enable to sufficiently obtain meiotic and developmental competence. However, those collected from small antral follicles (2-3mm) has been indicated that they difficultly achieve full maturation and reach to blastocyst stage. Hence, a sub-culture known as pre in vitro maturation (pre IVM) was applied to support such oocytes’ developmental competence. However, little is known about the correlation between antioxidant agent – L-ascorbic acid and pre IVM medium. Hence, this study was conducted to clarify such hypothesis. First, the cumulus oocyte complexes (COCs), obtained from small antral follicles were cultured in pre IVM treated with four different concentrations: 0µg/ml (AsA0), 25µg/ml (AsA25), 50µg/ml (AsA50) and 100µg/ml (AsA100). There were two control groups (negative control: oocyte from small follicle was not treated with pre IVM and positive control: oocyte derived from large follicles). The result showed that the increase in concentration of L-ascorbic acid induces the improvement of cumulus expansion in oocyte from small follicle comparable to the oocytes from large follicle, while without a pre IVM oocyte showed the poor cumulus expansion. In addition, oocytes from small follicle undergo pre IVM can reach to full maturation at metaphase II (MII) rate as same as oocyte from large follicle and lead to the high proportion of blastocyst comparable to the fully grown oocyte. Especially, the result showed that at the late blastocyst stage, AsA50 described the highest proportion and the similar percentage as fully grown oocytes, there were not significant differences between AsA0, AsA25, AsA50 and AsA100 in percentage of embryonic developmental rate. Study on the quality of embryo, we found that the group supplemented with 50µg/ml L-ascorbic acid can reach to blastocyst with high cell number like group of fully grown oocyte (53.33 % and 54.55% respectively). In conclusion, we found that the pre-IVM of oocytes from small follicles with 50µg/ml L-ascorbic acid, which reached the highest quantity and quality of blastocyst like fully grown oocyte. This induce a significant positive effect on the expression of epigenetic modification (both AcH3K9 and MeH3K9) in the bovine parthenogenetic blastocysts derived from small antral follicle.