Buffalos (Bubalus bubalis) Cloned by Nuclear Transfer of Somatic Cells1

Abstract

Cloning of buffalos (Bubalus bubalis) through nuclear transfer is a potential alternative approach in genetic improvement of buffalos. However, to our knowledge, cloned offspring of buffalos derived from embryonic, fetal, or somatic cells have not yet been reported. Thus, factors affecting the nuclear transfer of buffalo somatic cells were examined, and the possibility of cloning buffalos was explored in the present study. Treatment of buffalo fibroblasts and granulosa cells with aphidicolin plus serum starvation resulted in more cells being arrested at the G0/G1 phase, the proportion of cells with DNA fragmentation being less, and the number of embryos derived from these cells that developed to blastocysts being greater. In addition, a difference was found in the development of embryos reconstructed with fetal fibroblasts from different individuals (P < 0.001). Forty-two blastocysts derived from granulosa cells and fetal fibroblasts were transferred into 21 recipient swamp buffalos, and 4 recipients were confirmed to be pregnant by rectal palpation on Day 60 of gestation. One recipient received two embryos from fetal fibroblasts aborted on Day 300 of gestation and delivered two female premature calves. Three recipients maintained pregnancy to term and delivered three female cloned calves after Days 338-349 of gestation. These results indicate that buffalo embryos derived from either fetal fibroblasts or granulosa cells can develop to the term of gestation and result in newborn calves.

First cloned swamp buffalo produced from adult ear fibroblast cell

“…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. Serum starvation may often result in reduced cell survival and increased DNA fragmentation, which in turn cause subsequent high embryonic loss after nuclear transfer (Shi et al, 2007). In bovine nuclear transfer, Hill et al (2000) recorded 50% to 60% loss of cloned pregnancies by day 100; these rates are comparable to the loss rates encountered in the current study.…”

“…The natural delivery of the cloned calf following a normal gestation period (326 days) was within the range observed in normal breeding or natural insemination (325-332 days), as reported by Virakul (1987). However, differences in gestation period have previously been observed by Shi et al (2007) in cloned Chinese swamp buffalo, in which gestation periods for cloned pregnancies were found to be longer than for natural pregnancies. In the current experiment, birth weight of the cloned calf was at the higher end of the normal range (mean value, 26.68; range 15 to 41 kg), as established by Thevarnanoharan et al (2001).…”

“…Lu et al, (2011) noted that cloning of buffalo presents a greater challenge than other livestock. This may be attributable to their low fertility, as evidenced by delayed puberty, silent oestrous, long postpartum ovarian inactivity and lower oocyte recovery number from ovaries in comparison with cattle (Shi et al, 2007). Our first reports on swamp buffalo cloning showed that foetal fibroblast could be reprogrammed following transfer into recipient cytoplasts to produce cloned embryos up to blastocyst stage (Parnpai et al, 1999).…”

“…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. Of the three female cloned calves produced, only one survived.…”

Review on current application of genomic biotechnologies to improve livestock and livestock products

“…Today, there are estimated to be around 6000 farm animal clones worldwide (Plume, 2009). The cloning technology has been applied in the breeding of elite cattle (Kato et al,1998), goat (Baguisi et al,1999), pig (Polejaeva et al, 2000), horse (Galli et al,2003), buffalo (Shi et al,2007), camel (Wani et al,2010), Rabbet (Chesne et al, 2002) and other pet species like dog, cat, rat, ferret, mouse (Wakayama et al,1999;Roslin Institute online, 2003;Lee et al,2005and Shin et al,2002. Cloning is an asexual reproduction of genetically identical organism can be achieved by nuclear transfer (NT) or by embryo splitting (Abu et al, 2008).…”