The aim of study was the trial of deep conservation of breeding raccoon dog semen by use of EDTA diluent of different glycerol concentration. The semen was collected by manual method after earlier males accustomed to the room and the person who collected semen. All collected ejaculates were estimated macro- and microscopically. For further steps of dilution, equilibration and freezing ejaculates of volume above 0.3 cm, mobility of 40–60% of spermatozoa of progressive motion were qualified. Modified EDTA diluent was applied of the content 4% (RI), 6% (RII) and 10% (RIII) of glycerol as cryogenic factor. The process freezing and thawing of semen was controlled by mobility estimation as well as morphology of spermatozoa. In plasma of fresh and defrosted semen AspAT and acrosine activity was tested. From 66 collected ejaculates only 30 were qualified for freezing, what was 45.45% of all ejaculates. Sperm mobility in fresh semen was 50% average, but after defrosting participation of a mobile sperma tozoa decreased even up to 9.44%in semen with diluent RI with 4% glycerol addition. In this diluent participation of damaged spermatozoa increased from 35.5% to 53%. AspAT activity in fresh semen was 161.38 μIU•ml–1 average. After thawing, activity of the enzyme increased to 232 μIU•ml^–1 in RI, 372 μIU•ml^–1 in RII and only up to 170 μIU•ml^–1 in semen with diluent with 10% glycerol addition (RIII). Similar correlations were observed in acrosine activity.

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