328MEIOTIC COMPETENCE AND DNA FRAGMENTATION OF PORCINE OOCYTES FROM OVARIES STORED IN VARIOUS TEMPERATURES

Abstract

The aim of this study was to investigate the effects of storage of porcine ovaries at different temperatures before oocyte collection on the nuclear maturation and DNA fragmentation of cumulus-oocyte complexes (COCs). Ovaries were collected at a local abattoir and randomly kept in physiological saline at 4°C, 15°C, 25°C and 35°C. Ovaries were stored for 6 hours prior to follicle aspiration. After storage at each temperature (about 80 oocytes each group), COCs were fixed immediately after aspiration and stained by the terminal deoxynucleotidyl transferase (TdT) nick-end labeling (TUNEL) method to examine the DNA fragmentation under fluorescein microscope. To investigate meiotic competence of the oocytes, some COCs of each treatment group (about 100 oocytes each group) were matured in vitro for 45 hours in a modified North Carolina State University (NCSU)-37 solution supplemented with 10% (v/v) porcine follicular fluid, 0.6mM cysteine, 10IUmL−1 eCG and 10IUmL−1 hCG. After maturation culture, the cumulus cells were removed from COCs and fixed in acetic acid-ethanol (1:3, v/v) for 48–72h. The fixed oocytes were stained with acetic-orcein (1% orcein in 45% acetic acid) and examined under a phase-contrast microscope. Data were subjected to arc-sin transform before analyzing by ANOVA. The proportions of oocytes with DNA fragmentation increased with increasing storage temperature of ovaries (25.2% in 4°C, 31.8% in 15°C, 37.4% in 25°C and 54.7% in 35°C, respectively). There was no significant difference between the proportions of germinal vesicle breakdown (GVBD) of 25°C and 35°C storage groups (74.7 and 83.6%, respectively), but the proportions of 25°C and 35°C storage groups were significantly higher (P<0.05) than those of 4°C and 15°C storage groups (58.1 and 59.6%, respectively). The proportions of oocytes reaching metaphase II (MII) was significantly higher (P<0.05) in the 25°C storage group than in other groups (48.0% in 25°C v. 0% in 4°C, 0% in 15°C and 40.1% in 35°C). Moreover, none of oocytes in 4°C and 15°C storage groups reached MII. These results indicate that 25°C is the most suitable temperature for long-term storage of ovaries to maintain meiotic competence and prevent DNA fragmentation of porcine oocytes.