Influence Of Different Culture Systems On The Quality Of Somatic Cell Nuclear Transfer Embryos In Buffaloes
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Date
2010
Journal Title
Journal ISSN
Volume Title
Publisher
Tamil Nadu Veterinary and Animal Sciences University
Abstract
Out 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.
Description
Keywords
Buffalo, Cloning, Somatic cell nuclear transfer, Donor cell synchronization- activation, In vitro culture system, Epigenetic reprogramming