Direct Comparison of the Effects of Slow Freezing and Vitrification on Late Blastocyst Gene Expression, Development, Implantation and Offspring of Rabbit Morulae MD Saenz-de-Juano 1 , F Marco-Jimenez 1 , MP Viudes-de-Castro 2 , R Lavara 1 and JS Vicente 1 1 Instituto de Ciencia y Tecnolog  ıa Animal, Universidad Polite ´cnica de Valencia, Valencia, Spain; 2 Centro de Investigaci  on y Tecnolog ıa Animal, Instituto Valenciano de Investigaciones Agrarias, Segorbe, Spain Contents This study aimed to assess the effect of different cryopreser- vation procedures (slow freezing vs vitrification) on the gene expression in pre-implantation embryos and its implication in post-implantation embryo losses in rabbit. For this purpose, rabbit morulae were recovered at Day 3 of development, frozen or vitrified and transferred to recipients. Then, embryos were recovered on Day 3 post-transfer (Day 6 of development) or kept until the end of gestation. Apart from the gene expression analysis at Day 6, we also studied the pre- implantatory and foetal development ability of both cryopre- served embryo types by evaluating late blastocyst development at Day 6, embryo implantation at Day 11 post-transfer (Day 14 of development) and birth rate. We reported that slow freezing and vitrification have similar effects on embryo developmental ability till Day 6, but the distribution of losses changes during implantation and further development. These similarities at Day 6 of development were also reflected in gene expression patterns, and transcriptome analysis showed no differences between frozen and vitrified embryos. Our results confirm that vitrification provides better implantation and birth rates than slow freezing for rabbit embryos. As both the techniques are commonly used in human assisted reproduc- tion, further experiments must be conducted to clarify the causes that may hinder foetal development and their impact on adulthood. Introduction Cryopreservation of embryos is considered an impor- tant tool in both reproductive biotechnology of animal species and assisted reproduction in humans (Leibo and Songsasen 2002; Courbiere et al. 2013). In general, embryo cryopreservation procedure involves initial exposure to cryoprotective agents, cooling to subzero temperatures, thawing and return to physiological activity (Liu et al. 2012). At present, two major groups of methods for embryo cryopreservation can be defined: conventional slow freezing and vitrification, which mainly differ in the composition of cryoprotectants and cooling rates (Vajta and Kuwayama 2006). Con- ventional slow freezing was the first system described, which reported the first offspring produced by transfer of cryopreserved embryos (Whittingham et al. 1972). This method uses relatively low concentrations of ≤10% cryoprotective agents, cooling rates approximately 1°C/ min and warming rates approximately 250°C/min (Leibo 2012). However, the issue of ice crystal formation during cooling or warming steps becomes a major concern (Saragusty and Arav 2011). In 1985, Rall and Fahy (1985) introduced vitrification as a new method to cryopreserve mammalian embryos in the absence of ice. In contrast to slow freezing, vitrification methods use high concentrations of cryoprotective agents (30–40%), cooling rates much higher than 1000°C/min and very rapid warming rates (Leibo 2012). However, the highly concentrated solution of cryoprotective agents could be detrimental due to chemical toxicity or osmotic effect on cells (Vajta and Kuwayama 2006). Up to now, whether one technique is superior to the other is still a matter of controversy (AbdelHafez et al. 2010). However, comparisons of results have almost always demonstrated that vitrification is as good as or better than slow freezing (Leibo 2012) in terms of implantation and survival. Recently, we observed in rabbit embryos cryopreserved with slow freezing that the main losses of transferable embryos occurred before implantation and around placentation time (Saenz-de- Juano et al. 2012). In contrast, in the case of vitrified rabbit embryos, we showed how those embryos able to reach late blastocyst stage were also able to implant, but had a higher mortality rate from day 14 compared with fresh non-cryopreserved embryos, and a reduction in foetal development between days 10 and 14 of gestation (Vicente et al. 2013). The aim of this study was to evaluate and compare the effect of slow freezing and vitrification on global gene expression and in vivo development of pre-implantatory rabbit embryos. Material and Methods Unless stated otherwise, all chemicals in this study were purchased from Sigma-Aldrich Qu  ımica S.A (Madrid, Spain). Animals Rabbit does used as donors and recipients belonged to the New Zealand White line from the ICTA (Instituto de Ciencia y Tecnolog  ıa Animal) at the Universidad Polit ecnica de Valencia (UPV). All animals were han- dled according to the principles of animal care published by Spanish Royal Decree 53/2013. Experimental design Experimental design is shown in Fig. 1. Briefly, after artificial insemination, embryos were collected on Day 3 of development at morula stage; the embryos were frozen or vitrified and transferred to recipients after thawing/warming. To assess gene expression differences © 2014 Blackwell Verlag GmbH Reprod Dom Anim 49, 505–511 (2014); doi: 10.1111/rda.12320 ISSN 0936–6768