BackgroundEquine embryos >300 μm require puncture before vitrification. Protocols that do not require pre‐puncture would make vitrification easier and allow for its widespread use.ObjectivesTo design a successful vitrification protocol for embryos >300 μm without puncture as a pre‐treatment.Study designExperimental in vivo study.MethodsThirty‐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.ResultsPregnancy 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 limitationsLimited numbers and only one pregnancy was followed to term.ConclusionsEquine 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.

中文翻译：

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成功玻璃化冷冻大于 300 微米的马胚胎，无需穿刺或抽吸

背景>300 μm 的马胚胎需要在玻璃化冷冻前穿刺。不需要预穿刺的方案将使玻璃化冷冻更容易并允许其广泛使用。目的为> 300μm的胚胎设计一个成功的玻璃化冷冻方案，无需穿刺作为预处理。研究设计实验体内研究。方法将38个胚胎分开分为 3 组（G1：≤300 μm，n= 11；G2：>300–500μm，n= 20；G3：>500微米，n= 7）。使用人类玻璃化试剂盒对胚胎进行玻璃化冷冻。暴露于平衡溶液（ES；7.5% DMSO + 7.5% 乙二醇 [EG]，基础培养基 [BM] 为 M199 HEPES 缓冲培养基 [H199] + 羟丙基纤维素 + 庆大霉素）15 分钟后，胚胎暴露时间≤ 90 秒至玻璃化溶液（15% DMSO + 15% EG + BM 中的 0.5 M 海藻糖），加载到 Cryolock 上并投入 LN2。将 Cryolock 吸头浸入 1 mL H199 + 20% FBS + pen/strep + 1 M 蔗糖中，42°C 加热 1 分钟。然后将胚胎移至 0.5 M 蔗糖溶液中 4 分钟，然后在转移至受体之前置于 Vigro Hold 中 4 分钟。结果 G1 的妊娠率为 81.8% (9/11)，G1 为 80% (16/20)对于 G2，G3 为 0% (0/7)。存活的最大胚胎为 480 μm。主要限制数量有限，并且只有一次妊娠足月。结论 使用 ES 暴露时间较长的方案可以成功玻璃化≤480 μm 的马胚胎。这似乎不会对早期胚胎发育产生负面影响。