Improved functional oocyte enucleation by actinomycin D for bovine somatic cell nuclear transfer

Area of Science:

• Reproductive Biology
• Developmental Biology
• Animal Biotechnology

Background:

• Somatic cell nuclear transfer (SCNT) is a key technique for animal cloning but faces technical challenges, particularly in oocyte enucleation.
• Actinomycin D (AD) has been explored for oocyte enucleation in SCNT, but its toxicity and efficacy require optimization.

Purpose of the Study:

• To investigate and overcome limitations in functional oocyte enucleation using actinomycin D (AD) to improve bovine SCNT procedures.
• To assess the impact of AD treatment and subsequent spindle removal on early embryonic development and pregnancy rates in SCNT.

Main Methods:

• Bovine oocytes were treated with varying concentrations and incubation times of AD to determine optimal enucleation conditions.
• SCNT was performed using AD-treated oocytes, with and without spindle removal (NT(AD) and NT(AD+SR) groups), and compared to parthenogenetic (P(CTL)) and SCNT controls (NT(CTL)).
• Embryo development, caspase-3 activity, and pregnancy outcomes were evaluated for all groups.

Main Results:

• Improved AD enucleation protocols inhibited parthenogenetic development but restored cleavage rates in SCNT embryos.
• The NT(AD) protocol increased caspase-3 activity and did not improve blastocyst rates.
• Spindle removal post-AD treatment (NT(AD+SR)) normalized caspase-3 activity, improved embryo development, and resulted in mid-term pregnancies.

Conclusions:

• Optimized AD enucleation, coupled with spindle removal, effectively circumvents cellular roadblocks in early bovine SCNT development.
• This refined SCNT protocol shows promise for achieving pregnancies, although further research is needed to enhance blastocyst formation rates.