PARENT SESSION
Cryopreservation
529
EFFECT OF MATURATION STAGES ON MEMBRANE INTEGRITY OF BOVINE OOCYTES VITRIFIED BY THE OPEN-PULLED STRAW METHOD.
Brandão, Daniela1,2, Baraviera, Thais1, Rumpf, Rodolfo1, 1 2
ABSTRACT- Cryopreservation of bovine oocytes plays an important role in supporting and improving reproduction technologies. Besides that, it is fundamental to conserve genetic material of endangered species for future use. Many cytoesqueletical alterations are produced when oocytes are frozed, like membrane lesion. However, vitrification of bovine oocytes by open pulled straw (OPS) method has shown to produce less deleterious effects (Vajta et al, 1998). The aim of this experiment was to evaluate membrane integrity of oocytes vitrified by OPS in different stages of maturation. Ovaries from crossbred cows (B.indicus x B.taurus) were collected in slaughterhouse. The oocytes were matured in TCM199 supplemented with hormones and 10% fetal bovine serum (FBS) for 0, 8, 12 and 22h, when they were vitrified/warmed. The control group completed 24 hours of maturation to be evaluated. The vitrification consisted in a 25-30 sec exposure of the oocytes to TCM199-HEPES with 10% dimethilsulfoxide (DMSO), 10% ethylene glycol (EG) and 20% FBS. Then, they were exposed to TCM199-HEPES with 20% DMSO, 20% EG, 20% FSB and 0.5M of sucrose for 25 sec. The oocytes were loaded into the OPS straws and immersed into liquid nitrogen. They were warmed in TCM199-hepes with 20%FBS and 0.5M of sucrose solution at 38oC for 5 min, and then transferred to TCM199-hepes with 20%FBS and 0.25M of sucrose solution at 38oC for 5 min. The oocytes were immediately exposed to trypan blue solution (Sigma) for 5 min (Didion et al., 1993). The oocyte cytoplasm that turned to blue were considered as having membrane rupture or lesion. The percentages of membrane ruptured oocytes vitrified at 0, 8, 12, 22 hours and the control group were respectively 50.44% (116/230), 28.43% (56/197) , 16.84% (33/196), 25.14% (44/175) and 11.47% (18/157). The membrane rupture was significantly higher in oocytes vitrified at 0h (p>0.001) than in any other groups. Groups 8 and 22h were not different between each other (p<0.001). Only 12h group did not differ from the control oocytes (p<0.001). These results indicated that bovine oocytes may change the structure or the membrane composition during the maturation, showing different resistance to the vitrification procedure. It suggests that different freezing strategies and cryoprotectant solutions, according with maturation stage of the oocyte.
KEY WORDS: oocyte , vitrification, OPS, trypan blue