Post-vitrification survival and in vitro maturation rate of buffalo (Bubalus bubalis) oocytes: effect of ethylene glycol concentration and exposure time.

The present study was undertaken to investigate the effects of ethylene glycol concentration and time of exposure to equilibration solution on the post-thaw morphological appearance and the in vitro maturation rate of buffalo oocytes. Vitrification solution-I (VS-I) consisted of 4.5M ethylene glycol (EG), 3.4M dimethyl sulphoxide, 5. 56mM glucose, 0.33mM sodium pyruvate and 0.4% w/v bovine serum albumin in Dulbecco's phosphate buffered saline (DPBS), whereas vitrification solution-II (VS-II) contained 3.5M EG, with other constituents at same concentrations as in VS-I. The equilibration solutions-I and II were prepared by 50% dilution (v/v) of VS-I and VS-II, respectively, in DPBS. Prior to vitrification, the cumulus-oocyte complexes (COCs) were exposed to equilibration solution-I or II for 1 or 3min at room temperature (25-30 degrees C). Groups of four to five oocytes were then placed in 15microl of respective vitrification solution, and immediately loaded into 0. 25ml French straws, each containing 150microl of 0.5M sucrose in DPBS. The straws were placed in liquid nitrogen (LN(2)) vapour for 2min, plunged and stored in LN(2) for at least 7 days. The straws were thawed by keeping in warm water at 28 degrees C for 20s, and the oocytes were equilibrated for 5min in 0.5M sucrose for one-step dilution. The percentage of oocytes found to be morphologically normal varied from 89 to 96% for the two equilibration solutions and the two exposure times. Among the damaged oocytes, cracking of zona pellucida was the abnormality observed most frequently. The nuclear maturation rate of oocytes equilibrated in equilibration solutions-I and II for 1 (28 and 24%, respectively) or 3min (32 and 33%, respectively) did not differ significantly. These results show that it is possible to cryopreserve buffalo oocytes by vitrification using a combination of 3.5M EG and 3.4M DMSO with an exposure time of 3min.