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 10-6 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.
Highlights
Cryopreservation of equine embryos has been a challenge due to capsule development, mitotic activity and in particular, embryo size (Legrand et al, 2001; Stout, 2012)
All (12/12) of blastocysts exposed to ouabain or ouabainFL were positive for PI staining after incubation (Figure 3B)
Waelchli et al (1997) focused on trophoblastic vesicles, the authors cited that D-9 horse blastocysts partially collapsed after exposure to ouabain (10-6 M), with further re-expansion in ouabain-free medium in a pilot study, that was not observed in the present study
Summary
Cryopreservation of equine embryos has been a challenge due to capsule development, mitotic activity and in particular, embryo size (Legrand et al, 2001; Stout, 2012). Vitrification has become the preferred method to cryopreserve embryos of many species. The first foal born after vitrification was reported by Yamamoto et al (1982), with only 9% of frozen-thawed blastocysts resulting in live foals. Cryopreservation of equine embryos ≤ 300 μm of diameter resulted in satisfactory pregnancy rates (64 to 80%; Hochi et al, 1996). Pregnancy rates decreased when horse embryos > 300 μm were vitrified (MacLellan et al, 2001). When equine embryos were biopsied with a micromanipulator, they collapsed, but the embryos remained viable (Choi et al, 2010). The same authors subsequently evaluated effects of blastocoel cavity collapse in large
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