Abstract
The present study compares development of rabbit embryos generated using different oocyte activation protocols and reconstructed with embryonic or cumulus cells as nuclear donor. In vivo matured oocytes were collected from New Zealand White rabbits at 16 h after ovulation treatment and were activated atl8 h of post-ovulation treatment. The following schemes of oocytes activation were tested: 1) single electric pulse (EP, 3.2 kV/cm, 3x20 μs, 0.3 M mannitol)+5 min culture in the presence of 5 mM lonomycin, 2) single electric pulse (EP, 3.2 kV/cm, 3x20 μs, 0.3 M mannitol)+1 h culture in the presence of 2 mM 6-DMAP, and 3) three electric pulses 30 min apart. Cleavage rate, percentage of expanded and hatched blastocysts as well as total cell number of blastomeres of parthenogenetic embryos were significantly higher using either EP+6-DMAP or 3xEP schemes, comparing with EP+lonomycin. Development rate up to hatched blastocyst stage of cloned rabbit embryos using the EP+6-DMAP for activation of nuclei were 19% for embryonic cell nuclei and 36% for cumulus cell nuclei. The best activation protocol optimalized in this study was the combined treatment EP+6-DMAP, which may be potentially used for nuclear transfer protocol.
Highlights
Recent results from a number of laboratories indicate that one important factor affecting the developmental potential of activated and nuclear-transferred oocytes is the efficiency of oocyte activation (Ha et al, 1998; Inoue et al, 2002; Liu et al, 2002)
The subject of our study was to find an optimal protocol for rabbit oocytes activation using either embryonic or somatic cells as nuclear donor
Mature New Zealand White female rabbits were superovulated with single i.m. injection of PMSG
Summary
Recent results from a number of laboratories indicate that one important factor affecting the developmental potential of activated and nuclear-transferred oocytes is the efficiency of oocyte activation (Ha et al, 1998; Inoue et al., 2002; Liu et al, 2002). An oocyte can be activated beside a sperm, by artificial stimulus (electric pulse or chemical reagents) which induce a single intracellular calcium elevation. Aged oocytes can be activated more efficiency than younger oocytes by multiple electrical pulses, but the potential of activated oocytes to develop into blastocysts decreases with age (Collas and Robl, 1990). 6dimethylaminopurine (6-DMAP) was higher than the oocytes activated either with inositol 1,4,5-triphosphate alone or with multiple electrical pulses. Received July 31, 2003; Accepted January 27, 2004 with a combination of modified electric pulse conditions in the mannitol solution with Ca2+ could improve the activation and blastocyst development rates in rabbit oocytes (Ju et al, 2002). The subject of our study was to find an optimal protocol for rabbit oocytes activation using either embryonic or somatic cells as nuclear donor
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