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Bulletine of UASVM Animal Science and Biotechnologies
Year: 2010  |  Volume: 67  |  Issue: 1  |  Page No.: 489 - 489

Intravital Staining as Method to Assess Embryo Quality

Alexandra IVAN, Nicolae PACALA, Ada CEAN and Dorel DRONCA    

Abstract: During the last few years our group has focus on assessing mammalian embryos viability in order to improve methods currently used in different assisted reproductive techniques: embryo transfer procedures, in vitro embryo production, cultivation, and cryopreservation techniques. Besides morphometric characterization of in vivo and in vitro produced embryos, after embryo recovery/fertilization and during the in vitro cultivation we also applied different intravital staining tests. Fluoresceine diacetate (FDA) is a non fluorescent analog of fluoresceine, which can easily penetrate the cell membrane of the living cells and under cellular esterase activity is hydrolyzed to fluoresceine, which emit green fluorescence while exposed to a fluorescence source of 494-518 nm wave length. The FDA metabolism depends on the esterase activity and at the same time on the plasmatic membrane integrity. Propidium iodide (Pi) poses the capacity to penetrate the cell membrane of the nonviable cells, binds inside the DNA molecules and show a red fluorescence while the cells are exposed to a fluorescence microscope at 515-560 nm (Lakowicz, 2006). 0.5 mg/ml FDA were used together with 0.1 mg/ml Pi to form a mixture that allows us to observe at the same time both living and death cells within the embryo. The embryos recovered at 48 hours after hCG administration, were evaluated by morphological and intravital staining tests. By morphological criteria 18% of the embryos were evaluated as poor and very poor quality while applying the FDA -Pi, 13.79% of the embryos were scored as no viable, differences being statistically significant. After quality scoring the embryos were cultivated for 72 hours in M16 culture media to study their future developmental capacity. After 24 hours of in vitro cultivation on M 16 media the differences between the proportions of embryos evolving to the morula stage were distinct significant (χ2 test, p≤0.01), 61.3% for the control group comparing to 38.7% of the FDA and Pi group. After 48 hours of cultivation, the percentage of embryos, which developed was more reduce compared to the retarded embryos (χ2 test, p≤0.001), only for the control group embryos evolved to the hatched blastocyst stage (13.33%). For the FDA-Pi experimental group, embryos evolved in a small proportion to the expand blastocyst FDA and Pi (4.34%). Embryos developing up to blastocyst stage, indicates their ability to metabolize the staining dyes (Fluorescein diacetate), but the high percentage of retarded embryos shows a decreasing until stopping of developmental capacity after staining.

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