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The aims of the present study were to: (1) evaluate the effect of vitrification and warming on quality parameters and expression levels of pluripotency, apoptotic and stress genes in in vitro-produced (IVP) porcine blastocysts; and (ii) determine the correlation between these parameters. To this end, total cell number, DNA fragmentation, peroxide levels and the relative transcript abundance of BCL-2 associated X protein (BAX), BCL2-like 1 (BCL2L1), heat shock protein 70 (HSPA1A), POU class 5 homeobox 1 (POU5F1), superoxide dismutase 1 (SOD1) and superoxide dismutase 2 (SOD2) were analysed in fresh and vitrified IVP blastocysts. The results suggest that vitrification procedures have no effect on total cell number and gene expression of BAX, BCL2L1, SOD1 and SOD2 or the BAX:BCL2L1 ratio. Nevertheless, a significant increase in DNA fragmentation (2.9±0.4% vs 11.9±2.0%) and peroxide levels (80.4±2.6 vs 97.2±3.1) were seen in vitrified compared with Day 7 fresh blastocysts. In addition, after blastocyst vitrification, relative transcript abundance was downregulated for POU5F1 and upregulated for HSPA1A. Finally, there was a significant correlation of POU5F1 and HSPA1A with DNA fragmentation (POU5F1, r=-0.561; HSPA1A, r=0.604) and peroxide levels (POU5F1, r=-0.590; HSPA1A, r=0.621). In conclusion, under the conditions of the present study, vitrification and warming of IVP porcine blastocysts resulted in altered expression of POU5F1 and HSPA1A, but had no effect on BAX, BCL2L1, SOD1 and SOD2 expression.

The aims of the present study were to; (1) determine the effects of supplementation with two antioxidants during in vitro culture (IVC) on embryo development and quality; and (2) test the effects of adding the antioxidants to vitrification-warming media on the cryotolerance of in vitro-produced (IVP) porcine blastocysts. In Experiment 1, presumptive zygotes were cultured without antioxidants, with 50 µM β-mercaptoethanol (β-ME) or with 100 µM L-ascorbic acid (AC). After culture, blastocyst yield, quality and cryotolerance were evaluated in each treatment group. In Experiment 2, survival rates (3 and 24 h), total cell number, apoptosis index and the formation of reactive oxygen species (ROS) in blastocysts vitrified-warmed with 100 µM AC or 50 µM β-ME or without antioxidants added to the vitrification medium were compared. Antioxidant addition during IVC had no effect on embryo development, total cell number or the apoptosis index, and culturing embryos in the presence of β-ME had no effects on cryotolerance. In contrast, ROS levels and survival rates after vitrification-warming were significantly improved in embryos cultured with AC. Furthermore, addition of AC into vitrification-warming media enhanced embryo survival and embryo quality after warming. In conclusion, our results suggest that supplementing culture or vitrification media with 100 µM AC improves the quality and cryosurvival of IVP porcine blastocysts.

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