Vitrifikasi Blastosis Mencit dengan Metode Kriolupv ?O

I Wayan Batan; I Ketut Suatha; Wahono Esti PrasetyoningtyaserB; Nining Handayani; Ita Djuwita; Arief Boediono

I Wayan Batan
I Ketut Suatha
Wahono Esti PrasetyoningtyaserB
Nining Handayani
Ita Djuwita
Arief Boediono

Abstract

Cryopreservation is an ultra rapid freezing process to preserve tissue or organ. The studywas conducted to identify the effectiveness of cryoloop vitrification method and the viability ofembryos following vitrification. Embryos at blastocyst stage were vitrified by placing them inequilibration medium containing 10% ethylene glycol (EG) and phosphate buffer saline (PBS) wichsupplemented with 20% new born calf serum for 8-10 minutes. The blastocysts were then removedand put in vitrification medium (15% dimethyl sulfoxide, 15% EG, and 0.5M sucrose), and theprocess in the vitrifivcation medium not longer than 25-30 seconds. The blastocysts were immediatelytransferred to the vitrification medium film in the cryoloop and plunged into 100 ml liquid nitrogen.The warming process was done by immersing the cryoloop which carried the vitrified blastocstsinto PBS supplemented with 20% serum and 0.5M sucrose for 1 minute, and then removed to samesolution supplemented with 0.25M sucrose and 0.1M sucrose for 2 minutes respectively. Theblastocysts were washed 4 times in kalium simplex optimized medium (KSOM) and cultured indrops of KSOM in 5% CO2 incubator at 370C. The observations were done every 6 hours for 48hours using inverted microscope ( Olympus IX70 Japan). The viability of embryos was assessed onthe basis of the intact morphology, reexpansion of the blastosul, and the development of embryosinto advance stage. The results showed that 85.71% of vitrified embryos, developed into advancestages and 19% of them hatched. In conclusion the cryoloop can be used to vitrify the embryos.