Development of swamp buffalo (Bubalus bubalis) embryos after parthenogenetic activation and nuclear transfer using serum fed or starved fetal fibroblasts

Abstract

-The knowledge of oocyte activation and somatic cell nuclear transfer in the swamp buffalo (Bubalus bubalis) is extremely rare. The objectives of this study were the following: (1) to investigate the various activation treatments on the parthenogenetic development of buffalo oocytes, (2) to examine the events of nuclear remodeling and in the in vitro development of cloned buffalo embryos reconstructed with serum fed or starved fetal fibroblats, and (3) to investigate the in vivo development of cloned embryos derived from serum fed or starved cells after transfer into the recipients. The rates of cleavage and blastocyst development were found to be significantly higher (P < 0.05) when the oocytes were activated by the combination treatment of calcium ionophore (A23187) or ethanol followed by 6-DMAP than those activated by electrical pulses and 6-DMAP or other single treatments. Flow cytometric analysis revealed that the percentage in the G0/G1 phase in serum starved cells was significantly (P < 0.05) higher than that in serum fed cells (88.8 ± 6.2 vs. 68.2 ± 2.6). At 1 h post fusion (hpf), most of the transferred nuclei (71%) from serum fed cells did not change in size, and the nuclear envelope remained intact, whereas 29% underwent NEBD and PCC. When serum starved cells were used, 83% of the transferred nuclei underwent NEBD and PCC whereas 17% remained intact. The nuclear swelling and pronucleus (PN) formation were observed at 2-4 and 12 h post activation (hpa), respectively. The remodeled nuclei underwent mitotic division and developed to the 2-cell stage within 18-24 hpa. Fifty-five percent of oocytes reconstructed with serum fed cells were 2PN and 45% were 1PN, whereas 79% of the embryos reconstructed from starved cells were 1PN and 21% were 2PN. The percentage of blastocyst development of the embryos derived from starved cells was higher than that from the serum fed cells (35% vs. 21%, P < 0.05). Pregnancy was detected after the transfer of cloned blastocysts into the recipients but no recipients supported the development to term. The results of this work can be used to establish effective activation protocols for buffalo oocytes which can be used during nuclear transfer experiments.buffalo / oocyte activation / nuclear transfer / nuclear remodeling / somatic cell * Corresponding author: scykt@mahidol.ac.th 66 J. Saikhun et al.

First cloned swamp buffalo produced from adult ear fibroblast cell

“…The causes of abortion remain unclear, although incomplete genetic reprogramming or abnormal gene expression of nuclear transfer embryos may contribute to high abortion rates after embryo transfer (Saikhun et al, 2004). Incidence of loss between day 90 of gestation and calving was 43.7% for bovine adult somatic clones (Heyman et al, 2002), which was much lower than those encountered in the current study.…”

“…In this study, the 29% pregnancy rate at day 60 was higher than that reported by Saikhun et al (2004), who performed SCNT in synchronized buffaloes. In animals derived from cloning or in vitro culture procedures, a high incidence of placental abnormalities is found during both early and late gestation (Hill et al, 2000).…”

“…Moreover, Tasripoo et al (2007) showed that there was no difference in blastocyst formation among buffalo donor skin fibroblasts of a 6-month old calf, a 2-year old and a 4-year old buffalo. Furthermore, Saikhun et al (2004) found no significant difference in the development potential of embryos reconstructed with foetal, calf and adult fibroblast cells. Shi et al (2007) reported successful cloning of swamp buffalo by SCNT of foetal fibroblast and granulose cells.…”

Differential developmental competence and gene expression patterns in buffalo (Bubalus bubalis) nuclear transfer embryos reconstructed with fetal fibroblasts and amnion mesenchymal stem cells

“…Embryo production rate was examined under inverted microscope (Nikon Inc., Tokyo, Japan) to record the number of cleaved embryos at 48 h post-activation (h.p.a), percentage of embryos with 8-to 16-cells at 94-96 h.p.a and blastocyst formation at 168-192 h.p.a. Blastocysts were stained with Hoechst 33342 for 1 h and the total number of their nuclei was counted as described earlier by Saikhun et al (2004).…”

“…Blastocysts were stained with Hoechst 33342 for 1 h and the total number of their nuclei was counted as described earlier by Saikhun et al (2004).…”