40 IN VITRO SURVIVAL RATES OF IN VIVO- AND IN VITRO-PRODUCED BOVINE EMBRYOS CRYOPRESERVED BY SLOW CONTROLLED FREEZING OR VITRIFICATION

Abstract

Although slow programmable freezing is currently the standard method for bovine embryo cryopreservation, vitrification has become an alternative for in vitro-produced embryos. A study was designed to compare the in vitro survival rates of in vivo- and in vitro-produced bovine embryos with 1 of 2 commercially available methods of cryopreservation: slow freezing and solid surface vitrification. In vivo-produced Grade 1 blastocysts (n = 210) collected from superovulated donor cows 7 days post-insemination and in vitro-produced Grade 1 blastocysts (n = 122) from slaughterhouse oocytes, produced with the procedure described by Chaubal et al. (2007 Theriogenology 67, 719–728) were randomly allocated in 2 groups. Group 1 (slow freezing) embryos were exposed to 1.5 M ethylene glycol (ViGro EG; Bioniche Animal Health USA Inc., Pullman, WA, USA) for 5 min and loaded in 0.25-mL plastic straws. The straws were placed in a Freeze Control CL 5500 freezer (CryoLogic, Victoria, Australia) at –6.5°C, seeded and after 10 min of equilibration, cooled at –0.6°C min^–1 until –CE°C, before plunging into liquid nitrogen. Group B (vitrification) embryos were exposed to a AE% EG+0.BEM trehalose solution for A min and then into C0% EG+AM trehalose solution for C0 sec at room temperature to be vitrified using the CVM system (CryoLogic). The CVM used a cryohook and the solutions with the embryos are exposed to a metal solid surface cooled at –AIF°C. The vitrification solution was chosen after a toxicity test in which several EG and trehalose combinations were tested (Rodriguez Villamil et al. Ith IRAC Symposium, Argentina B0AA). After at least 1 wk of storage, embryos in the slow freezing groups were thawed in water bath at C0°C for AB s, placed in holding medium for E min and then cultured in SOF. Vitrified embryos were placed directly in a 0.BE M sucrose solution for E min (at CG°C) and then cultured in SOF medium. Re-expansion and hatching rates were evaluated at BD and GB h, respectively. Data was analyzed by nonparametric tests with type of embryo and cryopreservation procedure as main effects, using the software Infostat (UNC, Argentina, B0A0). In vivo-produced embryos had higher (P < 0.0A) re-expansion (AGI/BB0, HA% vs FI/ABB, EF%) and hatching rates (AEI/BB0, GB% vs EC/ABB, DC%) than in vitro-produced embryos, regardless of cryopreservation method. However, re-expansion (DE/FC, GA%) and hatching (CI/FC, FB%) rates were higher (P < 0.0A) with in vitro-produced vitrified embryos than in vitro-produced embryos in the slow freezing group (re-expansion: BD/EI, D0% and hatching: AD/EI, BD%). Although similar re-expansion rates (IC/AA0, HE% vs HF/A00, HF%) were obtained with in vivo- produced embryos cryopreserved by the 2 systems, hatching rates tended to be lower (P = 0.0I) with in vivo-produced embryos that were vitrified compared with slow freezing (GH/AA0, GA% vs HA/AA0, HA%). In conclusion, solid surface vitrification improved the cryosurvival rates of in vitro-produced embryos compared with the conventional slow, controlled freezing procedure.