Fluorescent Microscopy Analysis of Mammalian Oocyte Viability after Vitrification

Abstract

The paper reports the results of a fluorescent microscopy analysis of the viability of oocytes from cattle and pigs after vitrification. Oocytes were frozen in a vitrification media containing varying concentrations of cryoprotectors in several steps with subsequent vitrification. After cryobank storage for 14 days, experimental samples were thawed and oocyte viability was analyzed by oocyte morphology assessment and fluorescent microscopy. Two different kits were used to stain oocytes, one specific for necrosis/apoptosis (Propidium iodide/Alexa Fluor 488 Annexin) and the other specific for live/dead cells (Calcein-AM/ethidium homodimer-1). Fluorescent microscopy of porcine and bovine oocytes has demonstrated that the fluorescent dye Calcein-AM should be chosen to assess oocyte viability, since Propidium iodide and ethidium homodimer-1 do not represent the oocyte cell death. Therefore, Propidium iodide and ethidium homodimer-1 cannot serve as indicators of real oocyte death.