抄録
The present study was conducted to examine the meiotic competence and DNA damage of immature porcine oocytes following cryoprotectant exposure and vitrification. In the first experiment, porcine oocytes were collected from slaughtered ovaries and then exposed to four cryoprotectants [40% ethylene glycol (40%EG), 20% EG and 20% glycerol (20%EG/GLY), 20% EG and 20% propylene glycol (20%EG/PG), and 20% EG and 20% dimethyl sulfoxide (20%EG/DMSO)] for 1 min before in vitro maturation (IVM). In the second experiment, the porcine oocytes were vitrified with 40%EG, 20%EG/GLY, 20%EG/PG or 20%EG/DMSO before IVM. After cryoprotectant exposure or vitrification, the oocytes were cultured in the maturation medium for 44 h. As a control, the oocytes were cultured for 44 h without the treatments of cryoprotectant exposure and vitrification.The meiotic status and DNA fragmentation of the oocytes were examined using a combined technique for simultaneous nuclear staining and TUNEL by a modification of the procedures used by Otoi et.al.(1999). As a cryoprotectant, 20%EG/GLY was superior to 20%EG/PG and 20%EG/DMSO, with respect to the maturation to metaphase II (MII) and DNA fragmentation of oocytes. When the oocytes were vitrified and cultured, much less oocytes (5% to 9%) reached MII, irrespective of the kind of cryoprotectants. The percentages of DNA fragmentation in the oocytes with cryoprotectant exposure and vitrification were significantly lower than those of control fresh oocytes. These results indicate that the meiotic competence of porcine oocytes is damaged by the cryoprotectant exposure, and the competence of vitrified oocytes is not improved by the kind of cryoprotectants.
