Limits...
The aggregation of four reconstructed zygotes is the limit to improve the developmental competence of cloned equine embryos.

Gambini A, De Stefano A, Bevacqua RJ, Karlanian F, Salamone DF - PLoS ONE (2014)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Animal Biotechnology, Faculty of Agriculture, University of Buenos Aires, Buenos Aires, Argentina; National Institute of Scientific and Technological Research, Buenos Aires, Argentina.

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.

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Photographs of equine cloned foals derived from aggregated embryos.(A) Equine cloned foal derived from 4x experimental group, born on the 18th of September, 2013. (B) Equine cloned foal derived from 3x experimental group, born on the 12th of January, 2013.
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pone-0110998-g004: Photographs of equine cloned foals derived from aggregated embryos.(A) Equine cloned foal derived from 4x experimental group, born on the 18th of September, 2013. (B) Equine cloned foal derived from 3x experimental group, born on the 12th of January, 2013.

Mentions: Embryo transfer, pregnancy and survival rates for all experimental groups are shown in Table 4. Early pregnancy rates were higher when aggregated embryos were transferred; however, no statistical differences were found in pregnancy rates between non-aggregated and aggregated groups. Only pregnancies resulting from the aggregation of up to four embryos per well survived beyond the fifth month of gestation. One of the pregnant mares from the 3x experimental group showed clinical signs of Equine Metabolic Syndrome, dying in the last month of gestation. The cloned fetus presented normal vital parameters until the death of the mare. The two cloned foals obtained in this study derived from experimental groups 3x and 4x (Figure 4). Their gestation times were normal.


The aggregation of four reconstructed zygotes is the limit to improve the developmental competence of cloned equine embryos.

Gambini A, De Stefano A, Bevacqua RJ, Karlanian F, Salamone DF - PLoS ONE (2014)

Photographs of equine cloned foals derived from aggregated embryos.(A) Equine cloned foal derived from 4x experimental group, born on the 18th of September, 2013. (B) Equine cloned foal derived from 3x experimental group, born on the 12th of January, 2013.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4232247&req=5

pone-0110998-g004: Photographs of equine cloned foals derived from aggregated embryos.(A) Equine cloned foal derived from 4x experimental group, born on the 18th of September, 2013. (B) Equine cloned foal derived from 3x experimental group, born on the 12th of January, 2013.
Mentions: Embryo transfer, pregnancy and survival rates for all experimental groups are shown in Table 4. Early pregnancy rates were higher when aggregated embryos were transferred; however, no statistical differences were found in pregnancy rates between non-aggregated and aggregated groups. Only pregnancies resulting from the aggregation of up to four embryos per well survived beyond the fifth month of gestation. One of the pregnant mares from the 3x experimental group showed clinical signs of Equine Metabolic Syndrome, dying in the last month of gestation. The cloned fetus presented normal vital parameters until the death of the mare. The two cloned foals obtained in this study derived from experimental groups 3x and 4x (Figure 4). Their gestation times were normal.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Animal Biotechnology, Faculty of Agriculture, University of Buenos Aires, Buenos Aires, Argentina; National Institute of Scientific and Technological Research, Buenos Aires, Argentina.

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.

Show MeSH
Related in: MedlinePlus