Evaluation of Slow Freezing and Vitrification Methods of Cryopreservation in in vitro Produced Buffalo Embryos

Cryopreservation has been a revolutionary tool of assisted reproductive techniques to preserve valuable germplasm. The study was performed to measure the cleavage rate and the effect of cryopreservation techniques (slow freezing and vitrification) on in vitro produced buffalo embryos. Cumulus oocytes complexes were recovered by manual aspiration method. In vitro maturation (IVM), fertilization (IVF), and culture (IVC) were performed using commercially available Vitrogen (Brazil) media in benchtop incubator having 5% O2, 5% CO2, and optimum N2 with 90% humidity. Total 29 sessions were performed during November 2022 to June 2023 and 655 (23 ± 1.21 ovaries/session) ovaries were processed. Total 2515 follicles were observed including 1387 (55.15; 48.0 ± 1.81; 55.15%) small, 982 (34.0 ± 1.27; 39.05%) medium and 146 (5.0 ± 2.25; 5.8%) large follicles. Total 1679 COCs were recovered from 2515 follicles with recovery rate of 66.75%. Total 1640 COCs were used for IVM, IVF and IVC. After 72 h of the IVC 63.1 % (1015/1640) cleavage rate were observed and on day six after IVC 20 % (295/1640) blastocyst rate were observed. Blastocysts of grade I & II were randomly assigned either fresh, vitrified and slow freezing group. The re-expansion rate in frozen-thaw and vitrified-devitrified group blastocysts were observed at different time intervals. The total re-expansion rate of frozen-thawed BLs was 91.46%, which was significantly lower than 96% of vitrified-devitrified BLs. Further to study the hatching rate fresh (control) group was included and hatching were observed at 2, 12, and 24 hrs. Apoptosis rate were assessed using the TUNEL assay. TUNEL assay revealed a significant apoptosis in frozen-thaw blastocysts compared to vitrified-devitrified group (13.73 ± 0.67 vs 8.99 ± 0.71). Hence, vitrification is more competent method for cryopreservation of in vitro produced transferable buffalo embryos.