51 CASPASE-3 INHIBITOR Z-VAD-FMK ENHANCES CRYOTOLERANCE OF IN VITRO-PRODUCED BOVINE PRE-IMPLANTATION EMBRYOS

Abstract

In vitro-produced (IVP) bovine embryos are still less viable and resistant to cryopreservation than their in vivo counterparts. Cryopreservation induces cell degeneration through the apoptotic pathway in bovine oocytes and embryos (Men et al. 2003 Cryobiology 47, 73–81). Apoptosis can be prevented by inhibition of caspase activity, leading to improved cryosurvival in mammalian cells (Stroh et al. 2002 FASEB J. 16, 1651–3). Interestingly, cryotolerance of porcine embryos was improved by inhibiting apoptosis using a caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (Z-VAD-FMK) during vitrification and subsequent culture (Men et al. 2006 Theriogenology 66, 2008–16). Aim of this work was to evaluate whether cryotolerance of bovine IVP embryos may be improved by using Z-VAD-FMK during cryopreservation and post-warming in vitro culture. Abattoir-derived bovine oocytes (n = 753, over 4 replicates) were in vitro matured and fertilized according to standard procedures (Rubessa et al. 2011 Theriogenology 76, 1347–55). Twenty hours after IVF, presumptive zygotes were cultured in SOF medium at 39°C with 5% CO₂, 7% O₂, and 88% N₂. On Day 7, embryo yields were assessed and blastocysts (except the hatched blastocysts) were randomly divided in 2 groups: vitrification and post-warming culture in presence (n = 60) or absence (n = 54) of 20 µM Z-VAD-FMK. Vitrification was carried out by Cryotop in 16.5% ethylene glycol, 16.5% DMSO, and 0.5 M sucrose (Rubessa et al. 2011 Theriogenology 76, 1347–55). Blastocysts were warmed in decreasing sucrose solutions (0.25 M for 1 min and 0.15 M for 5 min) and cultured for 2 days. Resistance to cryopreservation was evaluated by assessing the survival rate, based on morphological criteria and hatching rate after 48 h culture. Furthermore, TUNEL staining was used to evaluate the total cell (TC) number and the apoptotic rate of vitrified blastocysts after 48-h post-warming culture. Differences between groups in survival and hatching rates after 48-h post-warming culture were analysed by Chi-squared test, whereas differences in TC number and in number and percentage of apoptotic cells were analysed by Student’s t-test. Inhibition of caspase activity induced by Z-VAD-FMK increased embryo cryotolerance, as indicated by higher survival (92.6 v. 55.0%; P < 0.01) and hatching rates (40.7 v. 23.3%; P < 0.05) after 48 h of post-warming culture. Furthermore, Z-VAD-FMK decreased both the average number (7.1 ± 0.6 v. 4.2 ± 0.3; P < 0.01) and the percentage (6.3 ± 0.6 v. 3.0 ± 0.2; P < 0.01) of apoptotic cells in blastocysts. No differences were recorded in TC number between groups (on average, 128.90 ± 1.6). These results suggest that addition of 20 µM Z-VAD-FMK during vitrification/warming and post-warming culture significantly inhibits apoptosis (DNA fragmentation) and improves the cryotolerance of IVP bovine embryos.