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Animal reproduction2020; 17(1); e20190079; doi: 10.21451/1984-3143-AR2019-0079

Inhibition of Na+, K+ -ATPase with ouabain is detrimental to equine blastocysts.

Abstract: Although equine blastocysts ≤ 300 µm in diameter can be successfully vitrified, larger equine blastocysts are not good candidates for cryopreservation. As Na, K-ATPase is involved in maintaining blastocyst expansion, perhaps inhibition of this enzyme would be a viable method of reducing blastocyst diameter prior to cryopreservation. Objectives were to evaluate effects of ouabain-induced inhibition of Na, K-ATPase in equine blastocysts. Sixteen mares were ultrasonographically monitored, given deslorelin acetate to induce ovulation, and inseminated. Embryos (D7 and D9) were harvested and Na, K-ATPase inhibited for 1 or 6 h by exposure to 10 M ouabain, either natural ouabain or conjugated to fluorescein (OuabainFL), during incubation at 37° C. Evaluations included morphometric characteristics (bright field microscopy) and viability (Hoescht 33342 + propidium iodide). Blastocysts incubated for 6 h in Holding medium + ouabain (n=3) had, on average, a 45.7% reduction in diameter, with adverse morphologic features and no re-expansion after subsequent incubation in Holding medium for 12 h. In subsequent studies, even a 1-h exposure to Ouabain or OuabainFL, caused similar reductions, namely 38.7 ± 6.7% (n=5) and 33.6 ± 3.3% (n=7) for D7 and D9 blastocysts, respectively. Ouabain binding was confirmed after OuabainFL exposition and all embryos (n=12) lost viability. We concluded that Na, K-ATPase inhibition with ouabain caused death of equine blastocysts and therefore was not a viable method of reducing blastocyst size prior to cryopreservation.
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Publication Date: 2020-01-22 PubMed ID: 32368275PubMed Central: PMC7189547DOI: 10.21451/1984-3143-AR2019-0079Google Scholar: Lookup
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  • Journal Article

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 examined if blocking Na, K-ATPase enzyme in equine blastocysts (early-stage embryos) using a compound called ouabain could reduce their size for more efficient cryopreservation. However, it was found that the process led to death of the blastocysts, making it an unsuitable method for size-reduction prior to freezing.

Study Design and Methodology

  • The study involved an experimental manipulation of 16 mares, each of which had been induced to ovulate using deslorelin acetate and inseminated.
  • Their embryos were harvested at two different developmental stages: days 7 (D7) and 9 (D9).
  • These embryos (blastocysts) were then exposed to 10 M ouabain in an attempt to inhibit the enzyme Na, K-ATPase. The exposure happened over two time intervals, 1 hour and 6 hours.
  • Some of the used ouabain was naturally occurring, while some was chemically conjugated to fluorescein (OuabainFL).

Measurements and Results

  • Evaluations were performed based on the embryos’ morphometric characteristics (analyzed through bright field microscopy) and their viability (evaluated using Hoescht 33342 and propidium iodide).
  • The results showed that the blastocysts underwent a significant reduction in size when incubated for 6 hours with ouabain (an average of 45.7% decrease). However, they displayed adverse morphologic changes, and did not re-expand after subsequent incubation in a neutral medium for 12 hours.
  • Further studies showed that even a shorter exposure to ouabain, of only 1 hour, caused a similar level of size reduction; 38.7 ± 6.7% and 33.6 ± 3.3% for D7 and D9 embryos, respectively.
  • All embryos lost their viability after OuabainFL exposure. The binding of ouabain to the embryos was confirmed through the fluorescence of OuabainFL.

Conclusion

  • The researchers concluded that the method of using ouabain to inhibit the Na, K-ATPase enzyme on equine blastocysts to reduce their size is not feasible for cryopreservation purposes, as it led to the death of the embryos.

Cite This Article

APA
do Nascimento AD, Marques JCC, Cezar ARR, Batista AM, Kastelic JP, Câmara DR. (2020). Inhibition of Na+, K+ -ATPase with ouabain is detrimental to equine blastocysts. Anim Reprod, 17(1), e20190079. https://doi.org/10.21451/1984-3143-AR2019-0079

Publication

Animal reproduction
ISSN: 1984-3143
NlmUniqueID: 101272804
Country: Brazil
Language: English
Volume: 17
Issue: 1
PII: e20190079

Researcher Affiliations

do Nascimento, Agnelo Douglas
  • Departamento de Medicina Veterinária, Laboratório de Reprodução Animal, Universidade Federal de Alagoas, Viçosa, AL, Brasil.
Marques, Juliana Carla Cavalcanti
  • Departamento de Medicina Veterinária, Laboratório de Reprodução Animal, Universidade Federal de Alagoas, Viçosa, AL, Brasil.
Cezar, Allan Rodolf Ribeiro
  • Departamento de Medicina Veterinária, Laboratório de Reprodução Animal, Universidade Federal de Alagoas, Viçosa, AL, Brasil.
Batista, André Mariano
  • Departamento de Medicina Veterinária, Universidade Federal Rural de Pernambuco, Recife, PE, Brasil.
Kastelic, John Patrick
  • Department of Production Animal Health, Faculty of Veterinary Medicine, University of Calgary, Calgary, AB, Canada.
Câmara, Diogo Ribeiro
  • Departamento de Medicina Veterinária, Laboratório de Reprodução Animal, Universidade Federal de Alagoas, Viçosa, AL, Brasil.

Conflict of Interest Statement

Conflict of interest: The authors have no conflict of interest to declare.

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Citations

This article has been cited 2 times.
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