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PloS one2014; 9(11); e110998; doi: 10.1371/journal.pone.0110998

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.
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Publication Date: 2014-11-14 PubMed ID: 25396418PubMed Central: PMC4232247DOI: 10.1371/journal.pone.0110998Google Scholar: Lookup
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Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

The research article focuses on the study of how the number of aggregated zygotes, reconstructed for cloning, affects the developmental competence of embryos in horses. The study’s findings suggest that combining up to four zygotes improves both in vitro and in vivo embryo development, with a decrease in development when five or more zygotes are used.

Research Objective and Methodology

  • Researchers sought to understand whether increasing the number of cloned zygotes aggregated for embryo development would improve the development of equine (horse) embryos both in vitro (outside the organism) and in vivo (within the organism).
  • The embryos used were “zona-free reconstructed embryos” (ZFREs) – embryos that have been segmented or reassembled after removing the protective layer (the zona pellucida) and were divided into four experimental groups.
  • Each of these groups represented varying numbers of ZFREs per well: group I included one ZFRE per well, group II included three, group III included four, and group IV included five.
  • Researchers measured the size of these embryos seven days into development. Then, the resulting blastocysts (early stage embryos) were either tracked for additional growth, examined for DNA fragmentation using the TUNEL assay, or transferred into synchronized mares for in vivo observation.

Key Findings

  • Researchers observed a higher rate of blastocyst formation on day seven in the group with four ZFREs per well as compared to the group with five ZFREs per well.
  • Non-aggregated embryos (those that were not combined from multiple zygotes) were smaller on day eight compared to the aggregated ones, implying that aggregation contributes to early growth. However, from day eight onwards, the growth rate was similar across all groups.
  • Apoptotic cells, or cells undergoing a programmed death, made up approximately 10% of all cells by day eight, with no discernible impact from embryo aggregation, suggesting that aggregation did not influence cell survivability.
  • Only pregnancies resulting from the aggregation of up to four embryos per well advanced beyond the fifth month of gestation. Two pregnancies developed into live foals, both from groups with three and four zygotes per well aggregation.
  • To summarise, the study demonstrated that the optimal number of zygotes for aggregation to improve equine embryo development, both in vitro and in vivo, is four. Development declined when five reconstructed zygotes were aggregated.

Cite This Article

APA
Gambini A, De Stefano A, Bevacqua RJ, Karlanian F, Salamone DF. (2014). The aggregation of four reconstructed zygotes is the limit to improve the developmental competence of cloned equine embryos. PLoS One, 9(11), e110998. https://doi.org/10.1371/journal.pone.0110998

Publication

PloS one
ISSN: 1932-6203
NlmUniqueID: 101285081
Country: United States
Language: English
Volume: 9
Issue: 11
Pages: e110998
PII: e110998

Researcher Affiliations

Gambini, Andrés
  • Laboratory of Animal Biotechnology, Faculty of Agriculture, University of Buenos Aires, Buenos Aires, Argentina; National Institute of Scientific and Technological Research, Buenos Aires, Argentina.
De Stefano, Adrian
  • Laboratory of Animal Biotechnology, Faculty of Agriculture, University of Buenos Aires, Buenos Aires, Argentina.
Bevacqua, Romina Jimena
  • Laboratory of Animal Biotechnology, Faculty of Agriculture, University of Buenos Aires, Buenos Aires, Argentina; National Institute of Scientific and Technological Research, Buenos Aires, Argentina.
Karlanian, Florencia
  • Laboratory of Animal Biotechnology, Faculty of Agriculture, University of Buenos Aires, Buenos Aires, Argentina.
Salamone, Daniel Felipe
  • Laboratory of Animal Biotechnology, Faculty of Agriculture, University of Buenos Aires, Buenos Aires, Argentina; National Institute of Scientific and Technological Research, Buenos Aires, Argentina.

MeSH Terms

  • Animals
  • Blastocyst / cytology
  • Cloning, Organism / methods
  • DNA Fragmentation
  • Embryo Culture Techniques
  • Embryo, Mammalian / embryology
  • Embryonic Development
  • Horses / embryology
  • In Situ Nick-End Labeling
  • Zygote / growth & development

Conflict of Interest Statement

The authors received funding from SIDUS Company. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials.

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