Veerapandian, C.Kathiresan, D.Subramanian, A.Parthiban, M.Reena, D.TANUVAS2016-05-192016-05-192010http://krishikosh.egranth.ac.in/handle/1/66142Out of 1371 ovaries collected from freshly slaughtered buffaloes a total of 7,227 oocytes were retrieved by slicing, with an average yield of 5.27 ± 0.68 per ovary. The maturation rates based on cumulus expansion and nuclear maturation in the Tissue Culture Medium 199 (TCM 199) media were significantly (P<0.01) greater than that in Synthetic Oviduct fluid (SOF) media (85.16 ± 3.14 vs. 69.09 ± 2.02 and 72.96 ± 3.27 vs. 50.80 ± 3.79 percent, respectively). In the experiment on the influence of various treatments to induce donor cells into G0/G1 phase (Gap phase 0/Gap phase 1) of cell cycle, fetal fibroblast subjected to serum starvation (SS), Confluence formation (CF) and Roscovitine treatment (RT) had fusion rates of (mean ± SE) 67.50 ± 4.79, 78.06 ± 1.94 and 57.03 ± 8.23 per cent, respectively. There was significant difference (P<0.05) in the fusion rates between treatment groups. Similarly with cumulus cells as donor cells there was significant difference (P<0.05) in the fusion rates between SS, CF and RT groups (80.82 ± 3.26, 59.49 ± 2.32 and 58.23 ± 0.60 percent, respectively). However, there was no significant difference observed between groups in both fetal fibroblasts and cumulus cells. Morula derived from cumulus cell synchronized with serum starvation only progressed into blastocyst at 3.90 ±2.55 per cent. In another experiment to compare two enucleation methods, the fusion rate was significantly (P<0.01) higher after physical aspiration than that after chemical enucleation (72.13 ± 2.53 vs. 55.36 ± 2.43 per cent). Cleavage and developmental rates of NT embryos were significantly (P<0.05) higher after physical aspiration than after chemical enucleation. Activation of fused couplets by ionomycin followed by subsequent administration of 6- Dimethyl aminopurine (6-DMAP) produced significantly (P<0.05) higher rates of cleavage and developmental stages of Nuclear Transfer (NT) embryos compared to activation by ionomycin + cycloheximide, strontium + 6- DMAP, strontium + cycloheximide, strontium + cytochalasin B. Ionomycin + 6- DMAP activated embryos alone progressed to blastocyst with a mean percentage of 1.78 ± 1.18. The cleavage rate of NT embryos was significantly higher (P<0.01) in G1/G2 systems (60.11 ± 1.28 per cent) when compared to co-culture, SOF and Potassium Simplex Optimization Medium (KSOM) (31.98 ± 1.93, 19.86 ± 1.98 and 12.22 ± 1.69, respectively). There was significant difference (P<0.05) in the developmental rates also among co-culture, KSOM and G1/G2 media. When two oxygen tensions for culture were compared, 5 per cent O2 tension had significantly (P<0.01) higher cleavage and developmental rates of NT embryos than 20 per cent O2 tension. Hatching occurred in one of the blastocyst cultured under 5 per cent oxygen level with hatching rate of 1.67 ± 1.67 percent. In another experiment on the use of the agents altering the levels of epigenetic modification of reconstructs, the treatment with 5-aza-deoxy cytadine (5-aza-dC), Trichostatin A (TSA) and combination of TSA and 5-aza-dC resulted in lower cleavage rates (mean ± SE) of 20.04 ± 2.64, 20.69 ± 2.16 and 38.33 ± 3.27 per cent, respectively when compared with control (47.41 ± 3.11). Morula derived from control and combination of TSA and 5-Aza-dC treatment groups only progressed into blastocyst at 2.08 ± 2.08 per cent in both. Similarly hatching occurred in one of the blastocyst cultured with combination of TSA and 5-aza-dC and control group. It is concluded that TCM-199 with supplements was more suited for in vitro maturation; cumulus cells synchronized by SS yielded more blastocyst: combination of ionomycin and 6-DMAP produced better activation: O2 tension at 5 percent gave better results than 20 and embryo culture was best supported by G1/G2 medium in the production of Somatic Cell Nuclear Transfer (SCNT) embryos.enBuffaloCloningSomatic cell nuclear transferDonor cell synchronization- activationIn vitro culture systemEpigenetic reprogrammingThesis