Somatic cells were isolated from cryopreserved semen of 4 buffalo bulls, 3 of which had died over 10 years earlier, and were established in culture. The cells expressed cytokeratin-18, keratin and vimentin indicating that they were of epithelial origin. The cells were used as nuclear donors for hand-made cloning for producing buffalo embryos. The blastocyst rate and quality, as indicated by apoptotic index, were comparable among embryos produced using cells obtained from fresh or frozen-thawed semen or those obtained from conventional cell sources such as skin. Examination of the epigenetic status revealed that the global level of H3K27me3 but not that of H3K9/14ac and H4K5ac differed significantly (P<0.05) among cloned embryos from different bulls. The relative mRNA abundance of HDAC1, DNMT1, P53 and CASPASE 3 but not that of DNMT3a differed in cells and in cloned embryos. Following transfer of 24 cloned embryos produced from fresh semen-derived cells to 12 recipients, one calf weighing 55 kg, which is now 6 months of age and is normal, was born through normal parturition. Following transfer of 20 embryos produced from frozen-thawed semen-derived cells to 10 recipients, 2 became pregnant, one of which aborted in the first trimester; the calf born was severely underweight (17 kg), and died 12 h after birth. The ability of cells derived from fresh and frozen-thawed semen to produce live offspring confirms the ability of these cells to be reprogrammed. Our findings pave the way for restoration of highly precious progeny-tested bulls, which has immense economic importance, and can also be used for restoration of endangered species.

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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In this study, we tested the effects of valproic acid (VPA), a known histone deacetylase inhibitor (HDACi), on the growth characteristics, apoptosis, and cell cycle stages distribution of donor cells, as well as cloning efficiency, embryo development, and histone methylation. Our results showed that treatment of donor cells with VPA (2.5 mM, 5.0 mM, 7.5 mM, or 10 mM) for 24 h resulted in altered cell proliferation, extent of apoptosis and necrosis, and cell cycle stage distribution, whereas no changes in cell viability and chromosomal complements were observed. Measurement of relative gene expression using real-time PCR of a few developmentally important genes in treated donor cells showed decreased expression of HDAC1 and increased expression of BAX (p<0.05). No change in relative expression of HDAC2 and Bcl2 was noticed. Treatment of donor cells with VPA for 24 h before electrofusion significantly (p<0.05) increased the blastocyst formation rate of somatic cell nuclear transfer (SCNT) embryos compared to the control embryos. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive nuclei in SCNT blastocysts derived from VPA-treated donor cells were significantly decreased compared to the control blastocysts (p<0.05). Immunolocalization studies revealed that the levels of histone H3 at lysine 9 (H3K9me3) were lower in VPA-treated donor cells derived cloned blastocysts than nontreated cloned embryos, and was at the level of in vitro fertilization (IVF) counterparts, although no effects of treatments were found in donor cells. Our study demonstrates that the use of VPA in SCNT has been beneficial for efficient reprogramming of donor cells. Its effect on histone methylation in cloned embryos correlates with their developmental potential and may be a useful epigenetic marker to predict the efficiency of SCNT.

We examined the effects of treating buffalo skin fibroblast donor cells with trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor, and 5-aza-2'-deoxycytidine (5azadC), a DNA methyltransferase (DNMT) inhibitor, on the cells and embryos produced by hand-made cloning. Treatment of donor cells with TSA or 5azadC resulted in altered expression levels of the HDAC1, DNMT1, DNMT3a, P53, CASPASE3 and CASPASE9 genes and global levels of acetylation of lysine at position 9 or 14 in histone 3 (H3K9/14ac), acetylation of lysine at position 5 in histone 4 (H4K5ac), acetylation of lysine at position 18 in histone 3 (H3K18ac) and tri-methylation of lysine at position 27 in histone 3 (H3K27me3). Moreover, global levels of DNA methylation and activity of DNMT1 and HDAC1 were decreased, while global acetylation of H3 and H3K9 was significantly increased in comparison to untreated cells. Simultaneous treatment of donor cells with TSA (50nM) and 5azadC (7.5nM) resulted in higher in vitro development to the blastocyst stage, reduction of the apoptotic index and the global level of H3K27 me3 and altered expression levels of HDAC1, P53, CASPASE3, CASPASE9 and DNMT3a in cloned blastocysts. Transfer of cloned embryos produced with donor cells treated with TSA led to the birth of a calf that survived for 21 days. These results show that treatment of buffalo donor cells with TSA and 5azadC improved developmental competence and quality of cloned embryos and altered their epigenetic status and gene expression, and that these beneficial effects were mediated by a reduction in DNA and histone methylation and an increase in histone acetylation in donor cells.

We compared handmade cloned (HMC) buffalo blastocysts produced from oocytes stained with Brilliant Cresyl Blue (BCB) and classified into those with blue (BCB + ) or colorless cytoplasm (BCB -). The blastocyst rate was higher ( p < 0.001) for BCB + than for BCB -oocytes (43.41 -2.54 vs. 22.74 -1.76%). BCB + blastocysts had inner cell mass (ICM) cell number, ICM-to-trophectoderm ratio, global level of H3K18ac, apoptotic index, and expression level of BCL-XL, but not that of CASPASE-3, similar to that of blastocysts produced through in vitro fertilization (IVF), which was higher ( p < 0.05) than that of BCB -blastocysts. The global level of H3K9me2, which was similar in BCB + and BCB -blastocysts, was higher ( p < 0.01) than that in IVF blastocysts. The expression level of OCT4 and SOX2 was higher ( p < 0.05) and that of GATA2 was lower ( p < 0.05) in BCB + than that in BCB -blastocysts, whereas that of DNMT1, DNMT3a, NANOG, and CDX2 was not significantly different between the two groups. The expression level of DNMT1, OCT4, NANOG, and SOX2 was lower ( p < 0.05) and that of CDX2 was higher ( p < 0.05) in BCB + than in IVF blastocysts. In conclusion, because BCB + blastocysts have better developmental competence and are closer to IVF blastocysts in terms of quality, epigenetic status, and gene expression than BCB -blastocysts, BCB staining can be used effectively for selection of developmentally competent oocytes for HMC.

The objective of this study was to explore the possibility of producing wild buffalo embryos by interspecies somatic cell nuclear transfer (iSCNT) through handmade cloning using wild buffalo somatic cells and domestic buffalo (Bubalus bubalis) oocytes. Somatic cells derived from the ear skin of wild buffalo were found to express vimentin but not keratin and cytokeratin-18, indicating that they were of fibroblast origin. The population doubling time of skin fibroblasts from wild buffalo was significantly (p < 0.05) higher, and the cell proliferation rate was significantly (p < 0.05) lower compared with that of skin fibroblasts from domestic buffalo. Neither the cleavage (92.6 ± 2.0% vs 92.8 ± 2.0%) nor the blastocyst rate (42.4 ± 2.4% vs 38.7 ± 2.8%) was significantly different between the intraspecies cloned embryos produced using skin fibroblasts from domestic buffalo and interspecies cloned embryos produced using skin fibroblasts from wild buffalo. However, the total cell number (TCN) was significantly (p < 0.05) lower (192.0 ± 25.6 vs 345.7 ± 42.2), and the apoptotic index was significantly (p < 0.05) higher (15.1 ± 3.1 vs 8.0 ± 1.4) for interspecies than that for intraspecies cloned embryos. Following vitrification in open-pulled straws (OPS) and warming, although the cryosurvival rate of both types of cloned embryos, as indicated by their re-expansion rate, was not significantly different (34.8 ± 1.5% vs 47.8 ± 7.8), the apoptotic index was significantly (p < 0.05) higher for vitrified-warmed interspecies than that for corresponding intraspecies cloned embryos (48.9 ± 7.2 vs 23.9 ± 2.8). The global level of H3K18ac was significantly (p < 0.05) lower in interspecies cloned embryos than that in intraspecies cloned embryos. The expression level of HDAC1, DNMT3a and CASPASE3 was significantly (p < 0.05) higher, that of P53 was significantly (p < 0.05) lower in interspecies than in intraspecies embryos, whereas that of DNMT1 was similar between the two types of embryos. In conclusion, these results demonstrate that wild buffalo embryos can be produced by iSCNT.

The application of cloning technology on a large scale is limited by very low offspring rate primarily due to aberrant or incomplete epigenetic reprogramming. Trichostatin A (TSA), a histone deacetylase inhibitor, and 5-aza-2'-deoxycytidine (5-aza-dC), an inhibitor of DNA methyltransferases, are widely used for altering the epigenetic status of cloned embryos. We optimized the doses of these epigenetic modifiers for production of buffalo embryos by handmade cloning and examined whether combined treatment with these epigenetic modifiers offered any advantage over treatment with the individual epigenetic modifier. Irrespective of whether donor cells or reconstructed embryos or both were treated with 50 nM TSA +7.5 nM 5-aza-dC, (1) the blastocyst rate was significantly higher (71.6 ± 3.5, 68.3 ± 2.6, and 71.8 ± 2.4, respectively, vs. 43.1 ± 3.4 for controls, p < 0.05); (2) the apoptotic index was lower (5.4 ± 1.1, 9.5 ± 1.0, and 7.4 ± 1.3, respectively, vs. 19.5 ± 2.1 for controls, p < 0.05) and was similar to that of in vitro fertilization blastocysts (6.0 ± 0.8); (3) the global level of H3K18ac was higher (p < 0.01) and that of H3K27me3 lower (p < 0.05) than in controls and was similar among all treatment groups; and (4) the expression level of epigenetic-(HDAC1, DNMT1, and DNMT3a), pluripotency-(OCT4 and NANOG), and development-related (FGF4) genes, but not that of SOX2 and CDX2, was similar among all treatment groups. These results demonstrate that similar levels of beneficial effects can be obtained following treatment of either donor cells or reconstructed embryos or both with the combination of TSA +5-aza-dC. Therefore, there is no advantage in treating both donor cells and reconstructed embryos when the combination of TSA and 5-aza-dC is used.