53 VITRIFICATION OF HANDMADE CLONED BUFFALO EMBRYOS USING ETHYLENE GLYCOL AND DIMETHYL SULPHOXIDE AND SUBSEQUENT EFFECT ON CRYOSURVIVAL AND APOPTOSIS

Abstract

The objective of the present work was to optimize concentration of cryoprotectants ethylene glycol (EG) and dimethyl sulphoxide (DMSO) to facilitate the vitrification of buffalo embryos produced by somatic cell nuclear transfer (SCNT). Cloned embryos were produced according to standardized protocols of our laboratory using handmade cloning (Shah et al. 2008). Three different concentrations of EG and DMSO (7.5, 10 and 15%, having 0.5 M sucrose in TCM-199 containing 20% serum) were selected in combination for vitrification of cloned blastocysts in French ministraws (0.25 mL). The numbers of cloned blastocysts vitrified for each concentration were 51, 51 and 52, respectively. The post-thaw viability was accessed by re-expansion rate of blastocysts after culturing in RVCL media (K-RVCL-50, Cook^® Australia, Queensland, Australia) for 18 to 24 h. On the basis of re-expansion rate, there was no significant effect of any selected concentrations (7.5, 10 and 15%) on post-thaw viability (25.33 ± 2.43%, 29.00 ± 2.52% and 30.83 ± 3.01%, respectively; P > 0.05). The effect of vitrification on apoptosis level was checked after 18 to 24 h post-thaw by TUNEL assay and the apoptosis index was calculated by dividing the total number of nuclei with DNA-fragmented positive nuclei of the respective blastocyst. We found that, 7.5%-group embryos resulted with a significantly higher apoptotic index (8.28 ± 0.57) than that of the 10 and 15% groups (5.09 ± 0.46 and 4.28 ± 0.24, respectively; P < 0.05). These results clearly indicate that a lower concentration of cryoprotectants (7.5%) increased the chance of apoptosis in blastocysts that were frozen-thawed. The quantitative expression of apoptosis-related genes (Bax, Bid, Mcl-1 and Bcl-xl) in all 3 treatment groups and fresh control embryos were determined by RT-qPCR. Three replications were performed and the mRNA level of each sample was normalized to that of glyceride-3-phosphate dehydrogenase mRNA level. Results of RT-qPCR were analysed using the 2^–ΔΔCT method to compare the relative transcriptional levels of the target genes in each group. The RT-qPCR data revealed that the 7.5% vitrified group embryos possessed high expression of pro-apoptotic genes (Bax and Bid) and lower expression of anti-apoptotic genes (Mcl-1 and Bcl-xl) in comparison to the 10 and 15% groups. However, there was no significant change in gene expression between the 10 or 15% groups in comparison with fresh non-vitrified embryos. Our results conclude that the best choice is to use 10 or 15% EG and DMSO cryoprotectants for in-straw zona-free cloned buffalo embryo vitrification. However, further experiments are needed to enhance survival after vitrification.

This work was supported by research grants from the National Agriculture Innovative Project (1(5)/2007-NAIP-2) to S. K. Singla.