EFFECT OF CRIOPRESERVATION AND BLASTOCELLECTOMY ON in vitro DEVELOPMENT OF BOVINE EMBRYOS PRODUCED in vivo

Abstract

Cryopreservation (CR) is a routine procedure in biology, livestock and medicine, and it is achieved with slow curve (CL) and vitrification (VT) procedures. The quality, stage of development and technique of CR alter the viability of post-thawed embryos. Blastocoelectomy (Blx) was developed to increase the survival rate of cryopreserved embryos. The hypothesis of this study was that the CR method by vitrification versus slow curve achieves a higher hatching rate (TE) in vitro of bovine blastocysts (BB) with or without blastocoelectomy. The objective was to determine the in vitro TE for BB cryopreserved by VT versus CL and with or without Blx. The experimental design was completely randomized with a 2?2 factorial arrangement, and the factors were CR and Blx. In 30 embryos Blx was performed and 30 other embryos remained intact; 50 % of each group was cryopreserved by CL and 50 % by VT (n?15, per treatment). Embryos cryopreserved by CL were exposed to 1.5 M ethylene glycol for 10 to 30 min and frozen at a speed of 0.5 °C min?1 in an automatic freezer. The VT was performed by introducing the embryos in equilibrium solution for 5 to 15 min, then they were transferred to four drops of vitrification solution and kept 5, 5, 10 and 10 s in each drop, respectively; then they were placed in Criotops? and immersed in liquid nitrogen. After thawing and heating, the blastocysts were cultured in IVC3? medium with 10 % of synthetic serum substitute for 72 h at 38.5 °C, 5 % CO2, 5 % air and 95 % humidity. The TE was analyzed by the ?2 method to determine the homogeneity between the proportions of the treatments. The effect of Blx on the survival rate and TE in vitro was not significant. VT as CR method of BB produced in vivo allows higher survival rate and better post-thawing hatching rate than the CL freezing method.