Murrone, Ombretta (2015) Vitrificazione dell'ovocita ovino: analisi delle modificazioni strutturali e molecolari. Doctoral Thesis.
In order to improve the vitrification efficiency of ovine oocytes, in the present study we compared two vitrification protocols (V1, V2). V1 protocol was designed to minimize toxicity and osmotic injuries of cryoprotectants (CPAs) by a dynamic exposure of oocytes to gradients of concentration of DMSO and EG, prior cryotop vitrification; V2 consisted in a two-step exposure to higher concentration of CPAs. Immature (GV, experiment 1) and in vitro matured oocytes (MII, experiment 2) of adult and prepubertal oocytes were vitrified by V1 and V2 protocols. After warming, oocytes were evaluated for survival, meiotic competence (GV), chromatin, spindle and actin configuration, gene expression, mitochondrial activity and ROS levels. In Experiment 3, the ability of Raman Microspectroscopy (RMS) to detect changes induced by V2 vitrification on the cortical F-actin of MII adult oocytes was evaluated. Results showed that V1 protocol was more suitable for adult GV and MII oocytes, in terms of survival rate, cytoskeleton integrity and ROS levels, whereas prepubertal oocytes were less affected by the type of the vitrification protocol. Moreover, GV oocytes of prebubertal and adult sheep had a lower resistance to vitrification than MII oocytes as demonstrated by the reduced meiotic competence. RMS analysis was able to discriminate oocytes with normal and abnormal F-actin and could represent an alternative analytical tool for investigating the biochemical modification of the oocyte.
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