In vitro nuclear progression of immature swamp buffalo oocytes after cryopreservation by vitrification method

A study was conducted to evaluate the effect of cryopreservation by vitrification method on the nuclear progression of immature swamp buffalo oocytes after in vitro maturation. Immature oocytes were aspirated from ovaries of slaughtered cows at various ages and reproductive condition and oocytes with compact cumulus cells and evenly granulated cytoplasm were selected and randomly divided into three groups. Group A oocytes were subjected to nuclear examination right after aspiration to provide baseline information on the nuclear stages of oocytes prior to cryopreservation and culture. Group B oocytes were cultured in vitro for 23-24 hours using Minimum Essential Medium supplemented with 10 percent Fetal Bovine Serum and antibiotics to serve as control. To evaluate the effect of cryopreservation, Group C oocytes were vitrified and stored in a liquid nitrogen tank for a minimum of 1 day after which were thawed and cultured in vitro as in Group B. To assess the nuclear progression, Group B and C oocytes were fixed with aceto-ethanol after in vitro maturation, stained with 1 percent Aceto-orcein and nuclear status was examined under the microscope. Nuclear stages of all the oocytes in each group were recorded and expressed on average and percentages. Results showed that 100 percent of the oocytes in Group A were at the germinal vesicle stage. After in vitro maturation, 86.66 percent of the oocytes (Group B) underwent germinal vesicle break down and are at initial stages of the second meiotic division. After cryopreservation (Group C), 93.5 percent of the oocytes were found at the germinal vesicle stage and 6.5 percent had undergone the germinal vesicle breakdown indicating a nuclear progression during in vitro culture. These results suggest that vitrification could be a potential technique for cyropreservation of buffalo oocytes and embryos but further modification and improvement of the present protocol to improve the present results is highly recommended