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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Photomicrographs of day 16 equine cloned embryo expression of TUNEL.(A, B, C) Day 16 Non-aggregated cloned equine embryo, 20x zoom. (D, E, F) Day 16 3x aggregated cloned embryo, 20x zoom. (G, H, I) Day 16 4x aggregated cloned embryo, 20x zoom. (J, K, M) Day 16 5x aggregated cloned embryo, 20x zoom.
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pone-0110998-g003: Photomicrographs of day 16 equine cloned embryo expression of TUNEL.(A, B, C) Day 16 Non-aggregated cloned equine embryo, 20x zoom. (D, E, F) Day 16 3x aggregated cloned embryo, 20x zoom. (G, H, I) Day 16 4x aggregated cloned embryo, 20x zoom. (J, K, M) Day 16 5x aggregated cloned embryo, 20x zoom.

Mentions: DNA fragmentation levels (mean ± SEM) of day 8 and day 16 cloned embryos of all experimental groups are shown in Table 3. There were no differences between groups, on day 8, in the levels of fragmented DNA, with an average of 10% positive TUNEL cells seen in all embryos (Figure 2). TUNEL-positive cells in day 8 embryos were: Group 1x (n = 6): 9.26%, 10.26%, 12.41%, 13.04%, 17.39% and 25.58%; Group 3x (n = 3): 8.97%, 9.43% and 10.29%; Group 4x (n = 5): 3.62%, 9.04%, 10.61%, 11.63% and 11.84%; and Group 5x (n = 2): 8.86% and 11.16%. There were no differences between groups on day 16 in the levels of fragmented DNA, with an average of 2.5% positive TUNEL cells seen in all cloned embryos (Figure 3). TUNEL-positive cell percentage of total cells counted per day 16 embryos and experimental group were: Group 1x (n = 3): 4.23%, 3.64% and 4.42%; Group 3x (n = 2): 3.35% and 0.33%; Group 4x (n = 3): 0.15%, 0.88% and 2.03%; and Group 5x (n = 3): 1.80%, 2.91% and 4.79%. Additional data are available in Figure S1.


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)

Photomicrographs of day 16 equine cloned embryo expression of TUNEL.(A, B, C) Day 16 Non-aggregated cloned equine embryo, 20x zoom. (D, E, F) Day 16 3x aggregated cloned embryo, 20x zoom. (G, H, I) Day 16 4x aggregated cloned embryo, 20x zoom. (J, K, M) Day 16 5x aggregated cloned embryo, 20x zoom.
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Related In: Results  -  Collection

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

pone-0110998-g003: Photomicrographs of day 16 equine cloned embryo expression of TUNEL.(A, B, C) Day 16 Non-aggregated cloned equine embryo, 20x zoom. (D, E, F) Day 16 3x aggregated cloned embryo, 20x zoom. (G, H, I) Day 16 4x aggregated cloned embryo, 20x zoom. (J, K, M) Day 16 5x aggregated cloned embryo, 20x zoom.
Mentions: DNA fragmentation levels (mean ± SEM) of day 8 and day 16 cloned embryos of all experimental groups are shown in Table 3. There were no differences between groups, on day 8, in the levels of fragmented DNA, with an average of 10% positive TUNEL cells seen in all embryos (Figure 2). TUNEL-positive cells in day 8 embryos were: Group 1x (n = 6): 9.26%, 10.26%, 12.41%, 13.04%, 17.39% and 25.58%; Group 3x (n = 3): 8.97%, 9.43% and 10.29%; Group 4x (n = 5): 3.62%, 9.04%, 10.61%, 11.63% and 11.84%; and Group 5x (n = 2): 8.86% and 11.16%. There were no differences between groups on day 16 in the levels of fragmented DNA, with an average of 2.5% positive TUNEL cells seen in all cloned embryos (Figure 3). TUNEL-positive cell percentage of total cells counted per day 16 embryos and experimental group were: Group 1x (n = 3): 4.23%, 3.64% and 4.42%; Group 3x (n = 2): 3.35% and 0.33%; Group 4x (n = 3): 0.15%, 0.88% and 2.03%; and Group 5x (n = 3): 1.80%, 2.91% and 4.79%. Additional data are available in Figure S1.

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