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

Abstract

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.

18 Embryo Aggregation in Pig Improves Cloning Efficiency and Embryo Quality

“…It is believed that for a successful cloning, the pattern of epigenetic modifications in the donor cells must be remodeled to be similar to the pattern present in the fertilized embryos [35].As a result, we performed embryo aggregation and obtained a significant increase in in vitro embryo development and cell number, 20% cleavage and a three-fold increase in blastocyst rate. Similar observations were also reported following embryo aggregation in domestic animals such as the pig following aggregation of 2 cells [33], or of 4 cells [11,14,36,37] and in the bovine [17], the horse [8,9] and the feline [10]. Increased cleavage, development rates and cell number proved that the medium used was adequate for normal development.…”

“…Embryo aggregation consists of placing more than one zona free embryos in the same well; some of the reported benefits in mammals are: improvement in embryo development in pig [6], equine [8,9], feline [10] and mouse [11], increasing the density of cells in the inner cell mass on cattle [12], blastocyst diameter in equine and in bovine [8,13], reduction in apoptosis in bovine and pigs [13,14,15], normalization of gene expression in bovine [15]and in some cases, it has also improved in vivo embryo development and early pregnancy rates in mouse [11], equine [8] and in cattle [13,16,17]. As a single clone may have epigenetic defects [15], embryo aggregation could compensate the reprogramming deficiencies through the interactions between blastomeres [11] or by paracrine pathways [11].…”

Embryos aggregation improves development and imprinting gene expression in mouse parthenogenesis

“…Recently, one study pointed that the aggregation of four embryos is the limit to improve the developmental competence of cloned equine embryo (Gambini et al . ). Whether or not that is also the limit for mouse still needs further study.…”

“…In addition, we tried to aggregate more than four parthenogenetic 4-cell embryos in a concave microwell (data not shown); however, numbers more than four could not aggregate to form one embryo. Recently, one study pointed that the aggregation of four embryos is the limit to improve the developmental competence of cloned equine embryo (Gambini et al 2014). Whether or not that is also the limit for mouse still needs further study.…”

Tiger, Bengal and Domestic Cat Embryos Produced by Homospecific and Interspecific Zona‐Free Nuclear Transfer

“…This last strategy consists on culturing several zona-free zygotes together that would be integrated into one single embryo. Embryo aggregation allows the complementation of clones with equal genomic characteristics but that have been reprogrammed differently, improving embryo development of clones and birth rates in different mammalian species (Zhou et al 2008;Ribeiro et al 2009;Gambini et al 2012Gambini et al , 2014.…”