Developmental potential of vitrified rabbit embryos

Abstract

The aim of this study was to examine how vitrification procedure can affect further development of preimplantation rabbit embryos. We evaluated post-thaw development and quality of the embryos following vitrification using morphological characteristics and fluorescent markers of the cell viability. The pronuclear stage eggs were flushed from the oviducts of the New Zealand rabbit females at 19-20 h post coitum (hpc) and randomly divided into two groups: first - intended for vitrification (n = 135) and second - non-vitrified (intact) control (n = 135) eggs. Then zygotes were cultured for 72 h, afterwards the embryos of the first group, which reached morula stage, were frozen by vitrification in EFS medium (ethylene glycol, Ficoll70, sucrose), whilst the embryos (morula) of the second (control) group continued in the culture until 120 h (expanded blastocyst stage). Then all the embryos including intact control (n = 270) were processed for total cell number, differential staining and cell death (apoptosis). Following thawing almost 73 % of embryos survived and developed to advanced blastocyst stage versus 96 % in the intact control. In regards to the embryo quality, in the vitrified embryos total cell number (117 ± 36.0) was significantly lower than in the intact control (135 ± 30.2). However, there were no significant differences between the vitrified and intact embryos in the proportion of ICM (inner cell mass cells) to the total cell number and in the embryo diameter. On the other hand, the incidence of dead cells (apoptotic index) was almost twice higher in the embryos subjected to cryopreservation (4.21 ± 1.85) compared to control (2.08 ± 0.50). However, this incidence of dead cells in vitrified embryos is still in the physiological range, and does not compromise further development. Our observation indicates that rabbit embryos after freezing have only slightly altered viability and quality. Therefore, vitrification techniques tested in our study can be used for cryopreservation of embryos of national rabbit breeds for the purpose of long-term storage of embryo samples in the animal gene bank.