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

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(-6) 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.