A comparison of the efficacy of three commercial human embryo vitrification kits for cryopreservation of in vivo produced equine embryos.

Abstract

Background: Different cryoprotectants can influence the ability of embryos to successfully survive vitrification and subsequent warming before transfer.

Objectives: To compare pregnancy rates for embryos ≤500 μm vitrified, without puncture or aspiration of the blastocoele cavity, with one of three commercial human embryo vitrification kits containing the same penetrating cryoprotectants (DMSO and EG) but varying in their non-penetrating cryoprotectants (NPCPAs; sucrose, trehalose, dextran serum supplement [DSS], and hydroxypropyl cellulose [HPC]).

Study design: In vivo experiments.

Methods: Embryos (n = 108) were vitrified using either a Kitazato (NPCPAs = trehalose, hydroxypropyl cellulose), Vit Kit Freeze (NPCPAs = sucrose, DSS), or Vit Kit Freeze NX (NPCPAs = trehalose, DSS) vitrification kit by exposing each embryo to kit-specific equilibration solution for 15 min, before the vitrification solution for ≤90 sec including loading onto a Cryolock device, which was capped and plunged into LN2. All embryos were warmed in the same media by placing the Cryolock tip into 1 mL of 1.0 M sucrose in a HEPES-based medium (1 min), followed by 0.5 M sucrose (4 min) and then commercial Holding medium (4 min) before transfer to a Day 6 recipient mare. For each kit, embryos were divided by size into three groups (G1 ≤ 300 μm; G2 > 300-400 μm; G3 > 400-500 μm; n = 8-14/group/kit).

Results: Pregnancy rates were equivalent for the Kitazato, Vit Kit Freeze, and Vit Kit Freeze NX kits for embryos in G1 (12/14 [85.7%] vs. 8/11 [72.7%] vs. 7/8 [87.5%], respectively, p = 0.63) and for G2 (10/12 [83.3%] vs. 10/11 [90.9%] vs. 9/11 [81.8%], respectively, p = 0.81). For G3 embryos, pregnancy rates were higher for the Kitazato versus either of the other kits (10/14 [71.4%] vs. 3/12 [21.4%] vs. 2/14 [14.3%], respectively, p = 0.003).

Main limitations: Limited numbers.

Conclusions: Different non-penetrating/extracellular cryoprotectants can influence the success of vitrifying equine embryos 400-500 μm. The combination of trehalose and hydroxypropyl cellulose appears to be beneficial in this respect.