METHOD FOR DETERMINING INTEGRITY OF CHROMATIN/DNA IN SPERMATOZOONS

摘要

FIELD: medicine.;SUBSTANCE: invention refers to medicine, namely to reproductive technologies, and can be used to determine the number of spermatozoons with fragmented DNA sections (IFn,%). That is ensured by using a fresh ejaculate, the smear of which is prepared for examination by liquefying the liquefied ejaculate with a phosphate-salt buffer with pH 7.2 and centrifugation at 2,400 rpm for 10 minutes, wherein precipitation is re-suspended in phosphate-salt buffer with pH 7.2 and concentration is adjusted to 20×10⁶ cells/ml; then smear is fixed on pre-cleaned defatted glass, then dried in air, obtaining a slide; said slide is fixed with alcohol fixing solution 96 %, and then dried in air and immersed in hydrolysing solution (0.1N HCl), followed by washing; staining slide in 0.05 % buffer solution of toluidine blue, washing in distilled water and drying in air; image cytometry is used to analyze the obtained results and to calculate the number of spermatozoa with fragmented DNA sections according to the presented formula, wherein the final result is the arithmetic mean of the results of at least three measurements, and if the IF_n is more than 30 % of the spermatozoons, the fertility is considered to be reduced.;EFFECT: method enables higher accuracy of determining the number of spermatozoons with fragmented DNA sections with simplification and shorter duration of the procedure.;7 cl