First steps of in vitro gastrulation in rabbit vitrified embryos

The in vitro rabbit embryo production and their cryopreservation methodologies such as vitrification generate less viable embryos, and occasionally, with significant differences from those that are not subjected to any treatment. Besides, in vitrified rabbit embryos little information is available about exactly when and where begin to emerge the first differences that finally result in foetal losses comparing with non-vitrified embryos.

The aim of this study was to evaluate the vitrification effects on the early in vitro gastrulation events.

After oviductal transfers of vitrified and non-vitrified embryos (control) in rabbit recipients, blastocysts from 144h (6-day-old) were recovered and cultured into TCM199 supplemented with rabbit homologous serum media for 48 hours. Gastrula stage and measures of perimeter and area of blastocyst and gastrula were noted. Moreover, eight independent pools consisting of six embryos each one were generated for each experimental group (control and vitrified) and total RNA was isolated to study the OCT4 gene expression.

Of 151 control and 164 vitrified morulae transferred, 69.5 % and 70.1 % developed in vivo to 6-day-old blastocyst respectively. After 24 hour of in vitro culture, 41.8 % of vitrified blastocyst had begun the neurulation (stage 5-) versus 22.8 % of control group. Nevertheless, the vitrified group showed the highest percentage of collapsed blastocyst at 48 hours (26.8 %). Non morphometric differences differences were observed in perimeter and area of blastocyst and gastrula between control and vitrified group at 0 and 24 hours. By contrast, perimeter and gastrula areas were slightly higher for the vitrified group than those for the control group at 48 hours of in vitro culture.

The study reveal the existence of the first morphological differences in vitrified blastocysts of 7 and 8-day-old, marked by a further development of gastrulation in the vitrified group.