This study investigated the effects of serum-starvation, total confluence, and roscovitine treatment on cell-cycle synchronization of buffalo ear skin fibroblasts to the G0/G1 stage and on the developmental competence of cloned embryos. Serum starvation of total confluence cultures for 24 h had a higher (p<0.05) proportion of cells at G0/G1 stage (94.4%) compared with serum starved cyclic and nonstarved confluent cultures (76.8 and 86.0%, respectively), whereas differences between cyclic cells with or without serum starvation were not significant. The proportion of cells at G0/G1 was higher (p<0.05) with 20 and 30 μM roscovitine treatment than that with 10 μM (94.4, 96.4, and 86.6%, respectively), which was similar to that for total confluence (86.0%). MTT assay showed that cell viability decreased as dose of roscovitine increased. The blastocyst rate was significantly higher (p<0.05) when nuclear transfer embryos were reconstructed using donors cells from total confluence, confluence serum starved, and roscovitine-treated (20 and 30 μM) groups (48.8, 48.9, 57.9, and 62.9%, respectively) compared to nontreated cyclic cells (20.2%). However, the cleavage rate and total cell number of cloned embryos were similar for all the groups. The number of ICM cells was improved by 30 μM roscovitine treatment (45.25 ± 2.34). The cryosurvival rate of blastocysts derived from cells synchronized with 20 or 30 μM roscovitine was higher compared to that for total confluence group (33.6, 37.8 vs. 23.8%). In conclusion, treatment with 30 μM roscovitine is optimal for harvesting G0/G1 stage cells for producing high quality cloned buffalo embryos, and that it is better than serum-starvation or total confluence for cell synchronization.

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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.