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Embryo aggregation has been demonstrated to improve cloning efficiency in mammals. However, since no more than three embryos have been used for aggregation, the effect of using a larger number of cloned zygotes is unknown. Therefore, the goal of the present study was to determine whether increased numbers of cloned aggregated zygotes results in improved  in vitro  and  in vivo  embryo development in the equine. Zona-free reconstructed embryos (ZFRE's) were cultured in the well of the well system in four different experimental groups: I. 1x, only one ZFRE per microwell; II. 3x, three per microwell; III. 4x, four per microwell; and IV. 5x, five ZFRE's per microwell. Embryo size was measured on day 7, after which blastocysts from each experimental group were either a) maintained in culture from day 8 until day 16 to follow their growth rates, b) fixed to measure DNA fragmentation using the TUNEL assay, or c) transferred to synchronized mares. A higher blastocyst rate was observed on day 7 in the 4x group than in the 5x group. Non-aggregated embryos were smaller on day 8 compared to those aggregated, but from then on the  in vitro  growth was not different among experimental groups. Apoptotic cells averaged 10% of total cells of day 8 blastocysts, independently of embryo aggregation. Only pregnancies resulting from the aggregation of up to four embryos per microwell went beyond the fifth month of gestation, and two of these pregnancies, derived from experimental groups 3x and 4x, resulted in live cloned foals. In summary, we showed that the  in vitro  and  in vivo  development of cloned zona-free embryos improved until the aggregation of four zygotes and declined when five reconstructed zygotes were aggregated.

The Aggregation of Four Reconstructed Zygotes is the Limit to Improve the Developmental Competence of Cloned Equine Embryos