75HIGHLY EFFICIENT AND RELIABLE CHEMICALLY ASSISTED ENUCLEATION METHOD FOR HANDMADE CLONING IN CATTLE AND SWINE

Abstract

In bovine and porcine nuclear transfer, most traditional enucleation procedures require potentially harmful chromatin staining and UV illumination. The purpose of our work was to find an efficient and reliable chemically-assisted procedure for enucleation connected to the handmade cloning (HMC) technique without chromatin staining. Slaughterhouse-derived oocytes were collected and matured in vitro. At 21 (bovine) or 43 (porcine) h after the start of maturation, cumulus cells were removed with vortexing and oocytes were further incubated in the maturation medium supplemented with 0.5μgmL−1 demecolcine for 2h. Subsequently, zonae pellucidae were digested with 2mgmL−1 pronase in the presence of 10% cattle serum (CS) for 6 to 8min and washed in HEPES-buffered TCM-199 medium and 20% CS. Bisection was performed in the same medium by hand under a stereomicroscope by using a microblade. A small membrane protrusion observable on the surface of oocytes was used as an orientation point. One-third of the cytoplasm connected to this protrusion was removed, and the cytoplasts and karyoplasts were collected separately. Bovine cytoplasts were used as recipients for HMC experiments (Vajta et al., 2003, Biol. Reprod. 68, 571–578) with fetal fibroblasts as donors, and reconstructed embryos were cultured for 7 days. In Experiment 1 (3 replicates), the possibility of oriented bisection at different time points was determined on a total of 225 bovine oocytes. At 5, 15, 25, 35 and 55min after the end of pronase digestion 64, 91, 93, 72 and 59% of oocytes had membrane protrusions (P<0.05 between all groups, SAS Genmod) illustrating the time-dependent manner of the protrusion. In Experiment 2, the efficiency and reliability of enucleation was measured. Bisection was performed between 5 and 35min after pronase digestion. Subsequently both supposed cytoplasts and karyoplasts were stained with Hoechst and investigated under UV light. In cattle (9 replicates), bisection was successfully performed in 94% (519/552) of oocytes, and 98% (507/517) of those bisected were enucleated, i.e. the chromatin was entirely in the presumptive karyoplast. In swine (3 replicates), 91% (302/331) of oocytes were successfully bisected and 95% (280/296) were enucleated. In Experiment 3 (cattle; 4 replicates), blastocyst per reconstructed embryo rates were 47% (139/293), illustrating the high developmental ability in vitro. Considering that no oocyte selection based on the presence of polar body was performed, the above system seems to be more efficient and reliable than other enucleation methods. Moreover, expensive equipment (inverted fluorescent microscope) and a potentially harmful step (staining and UV illumination) can be eliminated from the HMC without compromising the high in vitro efficiency.