28 Short equilibration improves vitrification of invitro-produced bovine embryos using VitTrans device

Abstract

For the successful application of vitrification technology to field conditions, the procedures for the warming and transfer of the cryopreserved bovine embryos should be as simple as possible. The device VitTrans, designed by our group, enables warming/dilution of embryos and their transfer directly to recipient females in field conditions (Morato and Mogas 2014 Cryobiology 68, 288). VitTrans vitrification protocol consists of an incubation in equilibration solution during 12min followed by an exposure of 40s to vitrification solution. However, there are other reports using similar vitrification devices where equilibration length is shorter than ours. This study aimed to improve VitTrans methodology by comparing two vitrification protocols: short equilibration (SE) and long equilibration (LE). A total of 63 invitro-produced Day 7 blastocysts (IETS stage code 7) were randomly placed in an equilibration solution with 7.5% ethylene glycol + 7.5% dimethyl sulfoxide in holding medium (tissue culture medium-199 HEPES + 20% fetal calf serum) for either 3min (SE) or 12min (LE). Then, blastocysts were transferred to vitrification solution (15% ethylene glycol + 15% dimethyl sulfoxide + 0.5M sucrose in holding medium) for 40s, loaded onto the VitTrans device, plunged into liquid nitrogen, and covered with a 0.5mL straw. Fresh nonvitrified blastocysts (n=30) were set as control. For warming, the VitTrans was quickly submerged into a water bath at 45°C, while a syringe containing 0.3mL of diluting solution (0.5M sucrose in holding medium) at 45°C was injected through the hollow of the device. Blastocysts were then transferred to synthetic oviductal fluid medium and cultured for 24h at 38.5% in a 5% CO2 and 5% O2 environment in a humidified atmosphere. Re-expansion rates were recorded 3 and 24h after warming. Blastocysts were fixed and stained with SOX2 (Invitrogen) for inner cell mass (ICM) count, TUNEL (Roche) for apoptosis index assessment, and DAPI (Vector Laboratories) for total cell count (TCC). Images were captured using a Leica TCS SP5 confocal microscope (Leica Microsystems) and examined with Imaris 9.2 software (Oxford Instruments). Blastocyst survival rates were compared between groups using chi-squared test. Blastocyst TCC, ICM count, and apoptosis indices were analysed using analysis of variance. Significance was set at P ≤ 0.05. No differences were observed in re-expansion rate at 3h postwarming (61.3 and 59.4% for SE and LE, respectively). However, significantly higher re-expansion rates were found after 24h of culture for the blastocysts of the SE group (74.2%) when compared with the blastocysts of the LE group (65.7%). Blastocysts vitrified using the LE protocol produced the lowest TCC (115±5.9; P ≤ 0.05), whereas TCC of the SE (152±9.7) and fresh control (138±8.6) treatments were similar. No differences were found in ICM count among groups. Nevertheless, apoptosis index was higher (P ≤ 0.05) in both vitrification groups when compared with fresh control. These results indicate that short equilibration vitrification not only improves VitTrans outcomes but adds efficiency by taking less time to perform. Supported by MCIU, Spain (Project AGL2016-79802-P and Grant BES-2017-081962).