Ceratonia siliqua (Carob) extract improved in vitro development of vitrified-warmed mouse germinal vesicle oocytes: assessment of possible mechanism

Oocyte banking is a vital step for safekeeping and spreading genetic resources of animals. It is also used for fertility preservation of human. Oocyte vitrification is closely related to the lower developmental competence which includes the cryo-injury arisen during vitrification. The aim of the present study was to evaluate the maturation, embryonic development and production of reactive oxygen species (ROS) of mice oocytes following the supplementation vitrification media with different concentrations of Ceratonia siliqua (carob) extracts. In this experimental study, germinal vesicle oocytes collected from 8 to 10 week-old female NMRI mice (30–40 gr) were randomly divided into six groups of vitrification media supplemented with 0 (control), 5, 10, 20, 30 and 50 µg/ml C. siliqua . After thawing, oocytes were put in an in vitro maturation medium (IVM) (α-MEM: Alpha Minimum Essential Medium). 3–4 and 24 h (hr) later, the oocyte nuclear maturity was checked. Standard in vitro fertilization was performed on the matured oocytes (MII), and embryonic development was followed. Extra- and intra-cellular ROS was measured in IVM medium after 24 h of oocyte incubation. The addition of 20 and 30 μg/ml C. siliqua extract to vitrification media improved normal morphology of warmed germinal vesicle (GV) oocytes, rate of germinal vesicle break down (GVBD), and metaphase 2 (MII) oocyte formation significantly ( p < 0.05). Fertilization rate, (embryonic development to 2 cells stage, 4–8 cells stage, and > 8 cells stage increased in the 30 μg/ml C. siliqua group significantly ( p < 0.05). Furthermore, supplementation of 30 μg/ml C. siliqua in vitrification media significantly decreased extra- and intra-cellular of ROS as well as embryonic fragmentation ( p < 0.05). In conclusion, supplementation of GV oocyte vitrification media with carob extract improved maturation, fertilization, and embryonic development rate and decreased extra- and intra-cellular ROS levels.