Tecniche innovative di crioconservazione : studio della metodica di solid surface vitrification (SSV)

Innovative cryopreservation techniques : study of the “Solid Suface Vitrification” (SSV) The purpose of the present study was to develope a promising cryopreservation procedure for biological samples, in particular for mammalian oocytes. A prototype of a Solid Surface Vitrification device, based of previous physical and computational data, was build for this purpose. Cat immature oocytes (GV stage) were placed in 4% ethylene glycol (EG) in TCM 199 plus 20% fetal bovine serum (FBS) at 37°C for 12–15 min, and then transferred to a vitrification solution (35% EG, 5% polyvinyl-pyrrolidone, 0.4 M trehalose inTCM 199 and 20% FBS). Oocytes were vitrified in microdrops (2ul) on a precooled (196°C) copper surface. The vitrified microdrops were stored in liquid nitrogen and were thawed after storage for 1–2 wk. Survival rate of vitrified–warmed oocytes was evaluated on the basis of ooplasm homogeneity, morphological appearence, nuclear integrity (Hoechst 33342 staining), zona pellucida intactness and esterase enzyme activity (diacetyl fluorescein staining). The results were compared with those obtained by a slow-freezeng procedure (paillettes). Cellular integrity and survival rate of GV obtained by SSV was comparable with that of the control procedure (67,9% and 67,9% vs. 70,7% and 74,1%), while the recovery rate was significantly lower, indicating that further improvements in thechnical aspects of the methodology are needed. Further investigations into the cryopreservation of feline oocytes by SSV may lead to more effective protocols.