Rabbit morula vitrification reduces early foetal growth and increases losses throughout gestation

Several studies have extensively examined structural and biochemical damage induced by cryopreservation that may lead to loss of rabbit embryo viability, but very little information is available on alterations in growth during gestation and at gene expression level. We started our work by comparing the distribution of losses of embryo and foetal development between control and vitrified rabbit morulae. Furthermore, data on foetal sack, foetal and maternal placenta and foetus size for 10-14 days of gestation were evaluated by ultrasonography. We reported that vitrification procedure causes detrimental effects on rabbit embryo and foetal development, with two major peaks of losses: one before the implantation (at day 6) and the other during the second part of gestation (after day 14). However, foetal loss may occur during the implantation process and placenta development, as there was a reduction in development of foetus produced from vitrified-warmed embryos between day 10 and 14 of gestation. For these reasons, using a recent microarray study performed in frozen-thawed rabbit embryos as a point of reference, we analysed the effects of vitrification procedure on the expression of 10 candidate genes in 6-day-old blastocysts obtained after vitrification and transfer. We observed that the relative expressions of mRNA transcripts from SCGB1A1, EMP1, ANXA3 and EGFLAM genes were significantly altered. This could help explain why a large number (29%) of vitrified embryos were successfully implanted but subsequently failed to develop to term. Further studies in subsequent embryo-foetal developmental stages, such as initiation of placenta formation, together with more sensitive high-throughput tools, should help us understand the deficiencies that hinder foetal development and identify the repairing mechanism employed by embryos to overcome vitrification effects.