Successful vitrification of equine embryos >300 microns without puncture or aspiration

Background Equine embryos >300 μm require puncture before vitrification. Protocols that do not require pre‐puncture would make vitrification easier and allow for its widespread use. Objectives To design a successful vitrification protocol for embryos >300 μm without puncture as a pre‐treatment. Study design Experimental in vivo study. Methods Thirty‐eight embryos were divided into 3 groups (G1: ≤300 μm, n = 11; G2: >300–500 μm, n = 20; G3: >500 μm, n = 7). Embryos were vitrified using a human vitrification kit. Following a 15 min exposure to equilibration solution (ES; 7.5% DMSO +7.5% ethylene glycol [EG] in a base medium [BM] of M199 HEPES‐buffered medium [H199] + hydroxypropyl cellulose + gentamycin), embryos were exposed for ≤90 s to a vitrification solution (15% DMSO +15% EG + 0.5 M trelahose in BM), loaded onto a Cryolock and plunged into LN2. Warming was undertaken by plunging the Cryolock tip into 1 mL of H199 + 20% FBS + pen/strep +1 M sucrose at 42°C for 1 min. The embryos were then moved to a 0.5 M sucrose solution for 4 min, then placed in Vigro Hold for 4 min prior to transfer to a recipient. Results Pregnancy rates were 81.8% (9/11) for G1, 80% (16/20) for G2, and 0% (0/7) for G3. The largest embryo to survive was 480 μm. Main limitations Limited numbers and only one pregnancy was followed to term. Conclusions Equine embryos ≤480 μm can be successfully vitrified using a protocol with a longer exposure time to the ES. This does not appear to have a negative effect on early embryonic development.