METHOD FOR DETERMINING CHROMATIN/DNA INTEGRITY IN SPERMATOZOA

摘要

The invention relates to reproductive technology and can be used for determining the number of spermatozoa having fragmented DNA regions (IFn, %). A smear of fresh ejaculate is prepared for examination by rinsing diluted ejaculate with phosphate buffered saline at pH 7.2 and centrifuging at 2400 rpm for 10 minutes, wherein the precipitate is re-suspended in phosphate buffered saline at pH 7.2 and the concentration is brought up to 20x106 cells/ml. The smear is fixed on a transparency then air-dried to produce a microscope slide; said slide is fixed with a solution of 96% ethanol, then air-dried, immersed in a hydrolysing solution (0.1N НСl) and rinsed; the slide is stained in a 0.05% buffer solution of toluidine blue, rinsed in distilled water and air-dried. Using image cytometry, the number of spermatozoa having fragmented DNA regions is calculated using the proposed formula. The arithmetic mean value of the results of not less than three measurements is taken as the final result, and if IFn is greater than 30% of the spermatozoa, fertility is considered to be reduced. This method increases the accuracy with which the number of spermatozoa having fragmented DNA regions is determined, while at the same time simplifying and shortening the procedure.