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 an equine cloned aggregated blastocyst in in vitro embryo culture beyond day 8.An equine cloned zona free blastocyst placed in a 100 µl drop of DMEM/F12 medium derived from the experimental group 3x during in vitro embryo culture from day 8 until day 16. (A) Day 9, 195.21 µm. (B) Day 10, 314.28 µm. (C) Day 11 444.75 µm. (D) Day 12, 498.76 µm. (E) Day 13, 672.17 µm. (F) Day 14, 1127.98 µm.
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pone-0110998-g001: Photographs of an equine cloned aggregated blastocyst in in vitro embryo culture beyond day 8.An equine cloned zona free blastocyst placed in a 100 µl drop of DMEM/F12 medium derived from the experimental group 3x during in vitro embryo culture from day 8 until day 16. (A) Day 9, 195.21 µm. (B) Day 10, 314.28 µm. (C) Day 11 444.75 µm. (D) Day 12, 498.76 µm. (E) Day 13, 672.17 µm. (F) Day 14, 1127.98 µm.

Mentions: In vitro growth rates of 14 cloned blastocysts (donor cell B) were determined daily. Growth patterns were similar between aggregated and non-aggregated groups. The number of embryos analyzed per group were: 1x: (n = 3), 3x: (n = 4), 4x: (n = 4) and 5x (n = 3). Each embryo within each experimental group derived from different replicates. Mean embryo sizes per day ±SD were: Day 8, 130.12 µm ±32.88; Day 9, 219.12 µm ±76.32; Day 11, 489.44 µm ±265.32; Day 12, 713.72 µm ±391.91; Day 13, 954.55 µm ±327.34; Day 14, 1664.87 µm ±713.54; Day 15, 2212.20 µm ±783.32 and day 16, 2677.18 µm ±977.43. Figure 1 shows an equine zona-free cloned blastocyst from the 3x experimental group during in vitro embryo culture.


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 an equine cloned aggregated blastocyst in in vitro embryo culture beyond day 8.An equine cloned zona free blastocyst placed in a 100 µl drop of DMEM/F12 medium derived from the experimental group 3x during in vitro embryo culture from day 8 until day 16. (A) Day 9, 195.21 µm. (B) Day 10, 314.28 µm. (C) Day 11 444.75 µm. (D) Day 12, 498.76 µm. (E) Day 13, 672.17 µm. (F) Day 14, 1127.98 µm.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0110998-g001: Photographs of an equine cloned aggregated blastocyst in in vitro embryo culture beyond day 8.An equine cloned zona free blastocyst placed in a 100 µl drop of DMEM/F12 medium derived from the experimental group 3x during in vitro embryo culture from day 8 until day 16. (A) Day 9, 195.21 µm. (B) Day 10, 314.28 µm. (C) Day 11 444.75 µm. (D) Day 12, 498.76 µm. (E) Day 13, 672.17 µm. (F) Day 14, 1127.98 µm.
Mentions: In vitro growth rates of 14 cloned blastocysts (donor cell B) were determined daily. Growth patterns were similar between aggregated and non-aggregated groups. The number of embryos analyzed per group were: 1x: (n = 3), 3x: (n = 4), 4x: (n = 4) and 5x (n = 3). Each embryo within each experimental group derived from different replicates. Mean embryo sizes per day ±SD were: Day 8, 130.12 µm ±32.88; Day 9, 219.12 µm ±76.32; Day 11, 489.44 µm ±265.32; Day 12, 713.72 µm ±391.91; Day 13, 954.55 µm ±327.34; Day 14, 1664.87 µm ±713.54; Day 15, 2212.20 µm ±783.32 and day 16, 2677.18 µm ±977.43. Figure 1 shows an equine zona-free cloned blastocyst from the 3x experimental group during in vitro embryo culture.

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