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SICHTAŘ JIŘI, SIMONIK ONDŘEJ, FOLKOVA PETRA, DOKOUPILOVA ADELA, RAJMON RADKO, SVOBODOVA IVONA, JEBAVÝ LUKAS. 2016. The Quality of Frozen-thawed Canine Semen With Respect to Semen Extender Composition and Sequence of Ejaculate Collection in Dogs. Acta Universitatis Agriculturae et Silviculturae Mendelianae Brunensis, 64(1): 169–175. The aim of this study was to evaluate the eff ect of clarifi ed egg yolk addition to semen extender, and the semen collection sequence on the quality of frozen-thawed semen in dogs. Semen was collected from 6 dogs in a time interval of 24 hours. As parameter of the quality of frozen-thawed (F-T) semen, the motility by computer assisted sperm analysis (CASA) and plasma membrane integrity by hypoosmotic swelling test (HOS) were evaluated. All kinematic parameters of sperm motility were higher in F-T samples containing the whole in comparison to the clarifi ed egg yolk. The sequence of semen collection aff ected sperm movement characteristics of native as well as F-T semen, but it was not possible to determine whether the fresh semen from the 1st or 2nd collection is of higher quality. All motility parameters of sperms frozen with extender containing the whole egg yolk were signifi cantly higher in the case of the 2nd collection. The situation was not so clear in the case of clarifi ed egg yolk addition, but the velocity values were higher in F-T samples from the 2nd collection. In contrast to proven diff erences in motility, the eff ect of the addition of clarifi ed egg yolk and the sequence of semen collection were not projected at all on the quality of plasma membrane of canine sperms evaluated by HOS test.