Chromatin and spindle organization of buffalo (Bubalus bubalis) in vitro matured oocytes following vitrification.

The aim of this work was to evaluate chromatin and spindle organization of buffalo in vitro matured oocytes after vitrification/warming by cryotop. In vitro matured oocytes were first exposed to 10 % ethylene glycol (EG) + 10 % dimethyl sulfoxide (DMSO) for 3 min, then to 20 % EG + 20 % of DMSO and 0.5 M sucrose, loaded on cryotops and plunged into liquid nitrogen within 25 sec. Oocytes were warmed into a 1.25 M sucrose solution for 1 min and then to decreasing concentrations of sucrose (0.62 M, 0.42 M and 0.31 M) for 30 s each. Two h after warming oocytes (n = 62) were fixed, immunostained for microtubules using a method previously described (Messinger and Albertini, 1991, J Cell Sci, 100, 289–98), stained for nuclei with propidium iodide and examined by fluorescence microscopy. Fresh in vitro matured oocytes (n = 54) were examined as control. Data were analyzed by Chi Square test. After vitrification the percentage of vitrified oocytes with abnormal spindle and chromatin configuration was higher compared with control oocytes (50 vs 9.2 %; P<0.01). The anomalies observed in vitrified-warmed vs control oocytes were: abnormal spindle with fragmented metaphase plate (58 vs 80 %), small spindle with contracted metaphase plate (39 vs 20 %) and abnormal spindle with normal metaphase plate (3 vs 0 %). It was demonstrated that oocyte vitrification in buffalo caused damage to the meiotic spindle and DNA fragmentation that may lead to aneuploidy incompatible with subsequent embryo development.