108 NEGLIGIBLE LEVEL OF DNA DAMAGE IN BOVINE OOCYTES VITRIFIED USING MINIMUM VOLUME METHODS

Abstract

A relatively high number of bovine cryopreserved oocytes analyzed by the comet assay (Men et al. 2003 Mol. Reprod. Dev. 64, 245) showed compromised DNA integrity. The DNA fragmentation (comet tails) was found in 29% of slow cooled oocytes, in 20% of oocytes vitrified in straws and in 24% of oocytes vitrified in open pulled straws (OPS). Present study used the comet assay to compare the DNA status of 151 in vitro matured bovine oocytes vitrified in straws, in OPS or in droplets. It was assumed that the droplet method (Papis et al. 2000 Theriogenology 54, 651), which has gentle pre-equilibration prior to vitrification, would offer better protection of DNA. OPS vitrification was performed using a solution consisting of 20% DMSO, 20% ethylene glycol (EG), and 0.5 M sucrose. For in-straw and in-droplet vitrification, VS14 (5.5 M EG and 1.0 M sucrose) solution was used. In these two methods pre-equilibration in 3% EG solution for 15 min was applied. Fresh oocytes exposed to 0.5 mM of hydrogen peroxide for 5 min served as the positive control. Fresh M II oocytes served as the negative control. The comet assay was performed according to the procedure of Men et al. (2003) with some modifications aimed at enhancing the sensitivity of the method. The zona pellucida was removed using 0.5% pronase solution, followed by placing of the oocytes in droplets of low-melting agarose on slides. These were subjected to overnight treatment in lysis buffer, followed by 40 min of DNA unfolding and 30 min electrophoresis. Following air drying, the slides were stained with DAPI fluorochrome and photographed. The pictures were saved as anonymous consecutive files to enable objective assessment. Of 119 vitrified oocytes, 112 (94%) were evaluated. The remainder were lost or displayed atypical pictures. The comets could not be analyzed with the Comet v.3.0 software, possibly due to the large size of each oocyte. Six main classes of comet tails were distinguished ranging from no tail (class 0) to heavy and long tail (class 5). Positive control oocytes displayed class 4 (36%) or 5 (64%) tails. Negative control oocytes formed class 0 (18%) to class 3 (47%) comet tails. The oocytes vitrified using minimum volume methods fell within the same range, with 80% and 76% of oocytes vitrified in droplets and OPS, respectively, forming class 1 or 2 tails. One OPS vitrified oocyte (2.2%) expressed a class 5 tail. A small but significant proportion of oocytes vitrified in straws (15.4%, P d 0.05, ANOVA) formed class 4 tails typical of positive control oocytes. In conclusion, in spite of pre-equilibration, a significant proportion of oocytes vitrified in straws contained detectable levels of DNA fragmentation, due probably to the lower cooling rate. The minimum volume protocols (the droplet and OPS methods) caused virtually no damage as assessed by the DNA comet assay. Results presented here differ from those reported previously. Reasons for differences remain to be established.