Metodologie innovative per la crioconservazione del germoplasma nella specie ovina

Aim of this work was to develop new methods for germplasm cryopreservation in the ovine species. The research was focused on new techniques and devices for the conservation of semen, tissues and embryos in an effective and cost-saving manner. The research was divided into three main experiments. In the former, the possibility of semen conservation after partial freeze-drying was tested; such approach would avoid the use of nitrogen and of the entire cold chain, with enormous advantages in terms of costs and feasibility. In the second and third experiments, a new micro-device (E.Vit), consisting of a 0.25 mm diameter straw with a 50 μm pore polycarbonate grid at one end, was used to create two protocols for vitrification of prepubertal animal testicular tissue and blastocyst stage embryos. The first experiment showed that ram semen maintains a good vitality after cryopreservation with low-toxic cryoprotectants and the partial freeze-drying technique suggest lyophilization as an alternative technique to classical freezing. E-Vit was successfully applied in both second and third experiments. Cell vitality and gene expression were evaluated in the vitrified-warmed testicular tissues, while the validity of the protocol for embryo cryopreservation was assessed in terms of post-warming survival and DNA fragmentation (TUNEL test). These new cryopreservation methodologies may significantly contribute to the improvement of reproductive technologies in both animal and human field.