Studies On The Nuclear Transfer Of Buffalo Somatic Cells

This study was undertaken to systematically examine factors affecting the nuclear transfer of buffalo somatic cells, so as to estabhsh a preliminary procedure for buffalo cloning Buffalo oocytes obtained from ovanes at slaughter were matured in vitro for 22?24h and then encleated in manipulation medium containing 5~ig/ml cytochalasin B Fibroblast and cumulus cells cultured in DMEM supplemented with 0 5%FBS (serum starvation)-. 0 1~ig/nil Aphidicolin (APD) +0 5%FBS for 2? days or 10%FBS after the 3? passages in DMEM+10%FBS, were transferred into enucleated oocytes by microinjection or electronic fusion The reconstructed embryos were activated by exposunng them to 5p~M ionomycin for 5mm and 2mm 6-DMAP for 3h Then the activated embryos were cultured in 301i1 drop of granulosa cell monolayers for 8?0 days, to evaluate their cleavage and embryomc development The cleavage rate of embryos reconstructed with fmbroblasts and cumulus cells pretreated with 0 1~gIml APD+0 5%FBS were significantly higher than the control group(p<0 01), however there was no significant difference in the percentage of cleaved oocytes developing to blastocysts When 1500 v Its/mm direct current field intensity was applied, 3 pulses resulted in a significantly higher fusion rate (72 04%) than 2 pulses, and a significantly higher cleavage rate (72 37%) than 4 pulse There was not significantly different in the cleavage rate and embryonic development among embryos denved from fibroblasts and cumulus cells pretreated with APD+0 5%FBS(63 06%Vs58 70% p>0 05) The cleavage rate of embryos reconstructed with cumulus cells by electrofusmon was significantly higher than that by nucromnjection (p<0 05), but there was not different in their proportion developing to blastocysts Sixty five to eighty five percent of cumulus cells at 3 and 6 passages, and fmbroblasts at 6 and 10 passages had normal karyotype, which did not show significant difference among them (p>0 05) When cumulus cells at 03, 04, G5 and G6 culture passage were used as donor cells for nuclear transfer, the cleavage rate was similar, but blastocyst development was significantly lower in 03 group compared with that in 04, G5 and G6 groups However, flbroblasts at G6, 07, G~ and 09 culture passages resulted in a smular cleavage rate and blastocyst development These results indicate that (1)Fmbroblasts and cumulus cells can be cultured up to 9 passages and keep relatively stable karyotype, (2) Using APD to treat donor cells prior to nuclear transfer can improve the efficiency of somatic cell nuclear transfer in buffalo but serum starvation is inefficient in our system, (3) The suitable electrofusmon parameters for nuclear transfer with cumulus cells are 10Ov/mm,15~is and 3 pulses , (4) Both of fmbroblast and cumulus cells can be used as the donor karyoplasts for nuclear transfer, and their efficiency was not influenced by the culture passages, (5) The development of NTE reconstructed by electrofusmon is higher than that by mlcromnjectmon, but there was no difference in the total efficiency between the two methods...