Rabbit embryo vitrification without animal products

Embryo cryopreservation often requires the use of animal derived products (such as bovine serum albumin (BSA) and fetal calf serum (FCS) ), which represent a sanitary risk, and contain undefined material that can considerably vary between batches, altering its thermodynamic properties. CRYO3 (Stem-Alpha, Saint-Genis-lu0027Argentiere France) is a chemically defined substitute without animal products, created for mononuclear cell cryopreservation. Recently, our research team successfully slow freezed rabbit embryos with a buffer medium composed of D-PBS (Dutscher, Brumath, France) supplemented with 20% of CRYO3 and 1.5M DMSO (Bruyere P et al. Plos One 8(8): e71547, 2013). Our objective was to compare three rabbit embryo vitrification buffer media: IMV holding medium (IMV, L’ Aigle, France): a commercial medium containing BSA (G1); D-PBS supplemented with 20% of CRYO3 (G2); and a CRYO3 medium (G3). All the media contained the same cryoprotectant composition. Rabbit New Zealand does (n = 12) were submitted to a superovulation treatment and collected embryos (n = 231) were randomly divided into three groups. After equilibration, embryos were exposed for 30 sec to a vitrification solution of G1 / G2 / G3 medium, containing 20 % Me2SO and 20 % EG, before being loaded to the hook at the end of a custom designed fibre called a Fibreplug™ (CVM Kit, Cryologic, Victoria, Australia) and vitrified by solid surface vitrification. Thawing was performed by immersing the end of the Fibreplug™ directly into a thawing solution (0.5 M sucrose, respectively), for 5 min, followed by three successive dilution baths. Embryos were cultured to the hatching stage in M199 medium (G1: 52, G2: 37 and G3: 71 embryos), supplemented with 10 % FCS (38.5°C, 5 % CO2). The survival rate after thawing (embryos with 20%) and smaller blastocysts were considered as damaged. The survival rate (G1: 87 %, G2: 81 % and G3: 96 % )) was significantly superior in the CRYO3 group (P u003c 0.05). No significant difference was observed regarding the blastocyst formation rate at 48 h (G1: 77 %, G2: 65 % and G3: 66 %) or the hatching rate (G1: 35 %, G2: 19 % and G3: 24 %).