29 The effect of vitrification at the immature stage on DNA methylation in porcine oocytes and its relevance to subsequent embryo development

Oocyte vitrification is an important approach for invitro gene banking of female germplasm; however, in pigs, it hampers embryo development. In cattle, vitrification at the MII stage was reported to alter epigenetic status in oocytes and even in subsequently developing embryos (Chen et al. 2016 Theriogenology 86, 868-878). The present study investigated the effect of vitrification at the immature stage of porcine oocytes on DNA methylation status and its relevance to subsequent embryo development. Immature cumulus–oocyte complexes were vitrified in microdrops and warmed (vitrified group) or treated with cryoprotectant agents (17.5% ethylene glycol + 17.5% propylene glycol, CPA group) by our method (Appeltant et al. 2018 Cryobiology 85, 87-94). Then they were subjected to IVM, parthenogenetic activation (PA), and embryo culture. From each batch, a group of oocytes was processed without treatment (control group). Oocyte survival and polar body extrusion were recorded after IVM. Cleavage and blastocyst developmental rates were recorded on Day 2 and 6 of culture, respectively (Day 0=PA). In each replication, DNA methylation was assayed in representative oocytes at the MII stage after IVM and in embryos at the 2- to 4-cell stage on Day 2 by immunostaining with 5-methylcytosine (5mC). Relative fluorescent intensity of 5mC in the chromatin was compared among groups. The experiment was replicated 3 times. Data were analysed by ANOVA. After IVM, there was no significant difference among the control, CPA, and vitrified groups in terms of the percentage of live oocytes (99.3, 96.4, and 94.0%, respectively) or polar body extrusion (88.6, 86.9, and 79.6%, respectively). After PA of oocytes with a polar body, there was no difference between the control and CPA groups in the percentage of cleavage (84.1 and 80.7%, respectively) or blastocyst development of cleaved embryos (63.3 and 79.3%, respectively). However, in the vitrified group, cleavage and blastocyst development rates (46.6 and 33.5%, respectively) were reduced (P<0.05) compared with the other groups. The 5mC fluorescence in the DNA of oocytes at the MII stage in the CPA and vitrified groups were similar and significantly lower than that in the control group (0.88±0.02, 0.87±0.001, and 1.0±0.02, respectively) but higher than that in the negative control processed without primary antibody (0.33±0.02). In the embryos at the 2- to 4-cell stage, 5mC fluorescence was not significantly different among the control, CPA, and vitrified groups (1.0±0.1, 0.99±0.1, and 0.96±0.1, respectively) but was significantly higher than that of the negative control (0.36±0.04). In conclusion, CPA treatment reduced DNA methylation levels in oocytes. However, it was restored during early embryo development and did not affect blastocyst development. The results suggest that reduced DNA methylation in vitrified oocytes is caused by CPA but it may not be responsible for their reduced ability to develop to blastocysts.