Highly efficient and reliable chemically assisted enucleation method for handmade cloning in cattle

Abstract

The purpose of the present study was to find an efficient and reliable chemically assisted procedure for enucleation related to the handmade cloning (HMC) technique. After in vitro maturation oocytes were incubated in 0.5 microg mL(-1) demecolcine for 2 h. Subsequently, zonae pellucidae were digested with pronase, and one-third of the cytoplasm connected to an extrusion cone was removed by hand using a microblade. The remaining two-thirds were used as recipients for HMC, and reconstructed and activated embryos were cultured for 7 days. The time-dependent manner of the development of extrusion cones, the efficiency (oriented bisection per oocyte; 94%), reliability (success per attempted enucleation; 98%), and the blastocyst per reconstructed embryo rates (48%) were measured. Ultrastructural analyses demonstrated that demecolcine treatment resulted in disoriented and haphazardly orientated microtubules. The general ultrastructure of the oocyte organelles, however, appeared to be unaltered by the treatments. Considering that no oocyte selection based on polar body presence was performed, this system seems to be more efficient and reliable than any other enucleation method. Moreover, expensive equipment (inverted fluorescence microscope) and a potentially harmful step (staining and ultraviolet illumination) can be eliminated from the HMC procedure without compromising the high in vitro efficiency.

The Effects of Demecolcine, Alone or in Combination with Sucrose on Bovine Oocyte Protrusion Rate, MAPK1 Protein Level andC-MosGene Expression Level

“…The enucleation can be accurately achieved based on the visible membrane protrusion. Vajta et al obtained high cloning efficiency with a 48% blastocyst rate by treating bovine oocytes with demecolcine [13]. Tani et al reported that treating bovine oocytes with demecolcine for over 30 min caused protrusions in the cell membrane and enhanced MPF activity in the oocytes during the process [14].…”

Chemically Assisted Handmade Enucleation of Porcine Oocytes

“…Several chemicals, including etoposide (Fulka and Moor, 1993;Elsheikh et al, 1997Elsheikh et al, , 1998Karnikova et al, 1998) and demecolcine (Gasparrini et al, 2003;Ibanez et al, 2003;Vajta et al, 2005), have been utilized to chemically induce or assist oocyte enucleation. Previously, attempts have been made to combine zonae-free manipulation with chemically induced or assisted enucleation in sheep and cattle (Peura, 2003;Vajta, 2005). According to Yin et al (2002a,b), the exposure of in vitro matured porcine oocytes to demecolcine results in an extrusion cone containing a condensed chromosome mass, which can be removed in TC.…”

Generation of handmade cloned embryos from adipose tissue derived mesenchymal stem cells in goat

“…Micromanipulator was routinely used to produce somatic cell cloned embryos in animals (Lagutina et al, 2007). However, the zona-free handmade cloning (HMC) technology is a preferred choice, with a higher success rate in producing cloned offspring in different species viz., horses (Lagutina et al, 2007), cattle Vajta et al, 2005), pig (Du et al, 2005) and buffalo (Shah et al, 2008). Handmade cloning (HMC) technique in goat is very significant on two accounts.…”