Vitrification of immature and matured bovine oocytes: effect of brilliant cresyl blue selection and hyaluronan addition
The aim of the present study was to evaluate the effect of brilliant cresyl blue (BCB) selection, the type of oocyte (immature or matured) and the use of hyaluronan in the vitrification solution on further embryo developmental competence. Oocytes (n = 1308) obtained from abattoir ovaries were classified by BCB stain. Control oocytes were maintained in holding media for 90 min and then subdivided to be placed into maturation media without any treatment or were vitrified. Immature or matured oocytes were vitrified by the solid-surface technique using two different vitrification solutions. VS1: composed of 10% ethylene glycol (EG) for 10 min followed by 20% EG + 0.2M trehalose for 30 sec and finally into 30% EG + 0.5M trehalose for 30 sec, or VS2 composed by 10% EG for 10 min, followed by 20% EG + 0.2M trehalose for 30 sec, and finally into 30% EG+ 0.5M trehalose + 0.1 g/ml hyaluronan for 30 sec. Oocytes were then loaded into Fyberplugs™ and vitrified. After one week, Fyberplugs™ were open and placed directly into (37°C) 0.5M sucrose solution for 5 min, then into 0.25M of sucrose for another 5 min and finally placed into maturation medium for in vitro production. Cleavage and development rates were examined on days 2 and 7 after fertilization, respectively. The blastocyst rate of vitrified oocytes selected as BCB + (5.5 ± 0.6%) were higher than those selected as BCB - (1.0 ± 0.4%) and those that were not selected by BCB (2.0 ± 1.1%; P < 0.001). Furthermore, immature vitrified oocytes had greater (P < 0.05) cleavage and blastocyst rates (44.8 ± 1.9% and 4.0 ± 0.6%) than matured vitrified oocytes (38.3 ± 2.8% and 2.5 ± 0.6%). Finally, the addition of hyaluronan to the vitrification solution had no significant effect on development rates. In conclusion, the selection of oocytes by BCB and the use of immature oocytes increase the development rates of vitrified-warmed oocytes.
BCB, cryopreservation, hyaluronan, oocyte selection.