The aggregation of four reconstructed zygotes is the limit to improve the developmental competence of cloned equine embryos.
Authors: Gambini Andrés, De Stefano Adrian, Bevacqua Romina Jimena, Karlanian Florencia, Salamone Daniel Felipe
Journal: PloS one
Summary
# Editorial Summary: Embryo Aggregation Limits in Equine Cloning Embryo aggregation—combining multiple cloned embryos into a single unit—has proven beneficial for cloning success in other mammals, yet the optimal number of equine embryos to aggregate remained unclear until this investigation. The research team cultured zona-free reconstructed equine embryos in four groups (1, 3, 4, or 5 embryos per microwell) and assessed blastocyst development rates, growth kinetics, cellular apoptosis via TUNEL assay, and pregnancy outcomes following embryo transfer to synchronised mares. Aggregating four reconstructed zygotes produced superior blastocyst rates on day 7 compared to the five-embryo group, whilst non-aggregated single embryos remained significantly smaller through day 8, though growth rates subsequently equalised across all groups; apoptotic cell levels remained consistent at approximately 10% regardless of aggregation strategy. Critically, only pregnancies derived from embryos aggregated in groups of up to four zygotes progressed beyond the fifth month of gestation, with two live foals successfully delivered from the 3x and 4x experimental groups, whilst five-embryo aggregations failed to establish viable pregnancies. These findings establish a practical ceiling for equine embryo aggregation in cloning protocols—four zygotes represent the optimum threshold beyond which developmental competence declines, offering valuable guidance for enhancing cloning efficiency in equine reproduction programmes.
Read the full abstract on PubMed
Practical Takeaways
- •For equine embryo cloning programs: optimize embryo aggregation at four zygotes per microwell to maximize development competence and pregnancy success.
- •Aggregating five or more cloned zygotes reduces developmental viability and pregnancy progression despite initial morphological improvements.
- •Early embryonic quality (apoptosis rates) is not the limiting factor—aggregation number itself determines developmental outcome in this system.
Key Findings
- •Aggregation of four cloned zygotes produced the highest blastocyst formation rate on day 7, with performance declining when five embryos were aggregated.
- •Aggregated embryos (3x, 4x, 5x) were significantly larger on day 8 compared to single embryos, though subsequent in vitro growth rates were similar among groups.
- •Only pregnancies from embryos aggregated in groups of up to four (3x and 4x) progressed beyond five months gestation, resulting in two live cloned foals.
- •Apoptotic cell rates remained approximately 10% across all aggregation conditions, independent of embryo number.