title: Simplified technique for differential staining of inner cell mass and trophectoderm cells of mouse and bovine blastocysts.
abstract: Histological staining and counting of blastocyst inner cell mass (ICM) and trophectoderm (TE) cells differentially with chromatin-specific dyes is a more accurate indicator of cultured blastocyst quality and normality than total cell number assessment. The aim of this study was to test the effectiveness of a simplified method of chemically-defined differential blastocyst staining. The TE of cultured mouse and bovine blastocysts of different developmental stages was stained when blastocysts were treated with a permeabilizing solution containing the ionic detergent Triton X-100 and the fluorochrome propidium iodide. Blastocysts were then incubated in a second solution containing 100% ethanol (for fixation) and the secondary fluorochrome bisbenzimide. Fixed and stained whole blastocysts were mounted and assessed for cell number using ultraviolet fluorescent microscopy. Using this method, in-vitro cultured mouse blastocysts (day 4.5) were shown to have an ICM:TE ratio of 1:2.63 with an average total cell count of 75.3 +/- 3. While day 7 and 8 in-vitro produced bovine blastocysts were shown to have an ICM:TE ratio of 1:3.42 and 1:3.36 with an average total cell count of 151.3 +/- 5.48 and 217.8 +/- 8.75 respectively. Blastocyst staining patterns indicate that this modified technique represents a simple and reliable alternative to current bichromatic blastocyst staining techniques for the differential assessment of cell numbers and may be useful for the assessment of blastocysts derived from in-vitro maturation, novel culture systems and advanced reproductive technologies such as cloning.
authors.affiliation: Centre for Early Human Development, Monash Institute of Reproduction and Development, Monash University, Melbourne, Victoria, Australia.
authors.forename: G. A.
authors.initials: GA
authors.lastname: Thouas
authors.forename: N. A.
authors.initials: NA
authors.lastname: Korfiatis
authors.forename: A. J.
authors.initials: AJ
authors.lastname: French
authors.forename: G. M.
authors.initials: GM
authors.lastname: Jones
authors.forename: A. O.
authors.initials: AO
authors.lastname: Trounson