Direct Warming of Vitrified In Vivo Equine Embryos

Vitrified in vitro-produced embryos can be successfully warmed in isotonic media at room temperature (RT; 22°C). However, this protocol has not been reported for in vivo embryos, which are more challenging to vitrify and warm. Study objectives were to see if vitrified in vivo embryos warmed in RT isotonic medium gave equivalent pregnancy rates to stepwise serial dilution warming, and if embryo size influenced the results. One hundred and seventeen embryos were divided into groups by size (G1:≤ 300 μm, n = 59; G2:> 300-400 μm, n = 33; G3:> 400-500 μm, n = 24) and vitrified using a commercial vitrification kit. Embryos were placed in an equilibration solution for 10 (embryos ≤ 300 μm) or 15 min (embryos > 300 μm) before being moved to a vitrification solution (≤ 90 s) and loaded onto a Cryolock prior to plunging into LN2. Warming was undertaken by placing the tip of the uncapped Cryolock into RT isotonic medium (n = 77; comprised of G1:n = 45; G2:n = 22; G3:n = 10) or by stepwise serial dilution (n = 40) with the initial 1 M sucrose solution at 42°C (G1:n = 14; G2:n = 12; G3:n = 14). Warmed embryos were transferred to Day 6 recipient mares and pregnancy rates compared between warming protocols. Ignoring embryo size, there was no difference in Day 14 pregnancy rates for vitrified embryos warmed in RT isotonic medium versus stepwise serial dilution (70.1%, 54/77 vs. 82.5%, 33/40, respectively). No statistical difference existed in pregnancy rates between warming protocols for G1 or G2 embryos (p = 1.0, p = 0.439, respectively), but for G3 embryos, the stepwise protocol results in significantly more pregnancies (p < 0.001). The largest embryo successfully warmed in RT isotonic medium was 390 μm. Whereas for the stepwise protocol, the largest embryo was 480 μm. Direct warming in RT isotonic medium is a suitable protocol for warming vitrified embryos ≤ 390 μm, although the decline in pregnancies at the upper limit of G2 would suggest that clinically this methodology is suitable for embryos ≤ 360 μm.