The aim of this study was to investigate the influence of progesterone, cholesterol and calcium (Ca(2+)) in an egg-yolk-containing extender on capacitation and acrosome reactions (AR) of diluted canine spermatozoa during 4 days of cooled-storage. For this purpose, we first investigated the effect of supplementation of a Tris-citrate-fructose buffer (TCF) with progesterone in a final concentration of 0.1, 0.2 and 1.0 microg progesterone/ml TCF-diluted semen. We then compared the effects of TCF and the same buffer-containing 20% egg yolk (TCF-EY). In egg yolks and the TCF-EY, progesterone was measured by enzyme immunoassay, cholesterol by enzymatic colorimetry and Ca(2+) by flame atomic absorption spectrophotometry. For both experiments, ejaculates from eight dogs were used. For the comparison of diluents, one ejaculate was divided and one half diluted with TCF, the other with TCF-EY. One half of each TCF- and TCF-EY-diluted sample was evaluated immediately (D1), the other after storage for 4 days at +4 degrees C (D4). In diluted semen, motility and viability were measured by a computer assisted sperm analyzer (CASA; Sperm Vision, Minitüb, Germany), capacitation and AR were evaluated with a modified chlortetracycline assay (CTC) and the AR additionally by flow cytometry. Results: Supplementation of progesterone revealed, that between D1 and D4, total and progressive motility decreased with all progesterone concentrations, while viability as well as percentage of capacitated and acrosome reacted spermatozoa stayed constant. Progesterone-, cholesterol- and Ca(2+) concentrations in egg yolks were 524.8+/-131.4 ng/g, 13.9+/-2.03 mg/g and 1.27+/-0.17 mg/g, respectively. In the TCF-EY-diluent, the respective values were 210.9 ng/g, 2.52 mg/g and 1.1mg/g. In TCF-semen, at D1, motility and viability were significantly higher than in TCF-EY-samples (p<0.05), however at D4, no significant differences were detectable. Further, in TCF-semen, percentages of spermatozoa with intact membranes decreased significantly (p<0.05) and capacitated spermatozoa increased (p<0.05), which was not seen in TCF-EY-samples. In all samples, low percentages of AR were detected and after 4 days, the highest value of AR in TCF-EY-samples was 5.3% on average, as detected by flow cytometry. We therefore conclude that progesterone from egg yolk in routine extenders does not substantially influence semen longevity or AR of canine semen during cold-storage for 4 days. In contrary, egg yolk seems to prevent a significant increase in capacitated spermatozoa.