Contents This study aimed at comparing in vitro, ultra-heat-treated (UHT) skim milk and INRA-96 (R)-based extenders supplemented or not with 5% egg yolk and/or 2% glycerol on sperm quality parameters along 72 h of preservation at 5 degrees C, using a factorial design. Semen from six healthy mature Merino rams was pooled and extended in each medium using a split sample procedure (six replicates) and chilled. Subjective motility (SM) (%), membrane integrity (MI) (%) and uncapacitated spermatozoa (US) (x106 spermatozoa/AI dose) were used to assess the semen quality at 0, 12, 24, 48 and 72 h of preservation. UHT-based extenders yielded better (p < 0.05) SM and MI than INRA-96 (R)-based extenders (59.7% vs 57.9%; 60.2% vs 55.8%, respectively) but similar numbers of US (64.2% vs 62.3 x 106 sperm/AI dose, respectively) along the preservation time. Egg yolk-glycerol or just egg yolk as additives improved (p < 0.05) the results compared with the base extenders without additives or just with glycerol. The sperm parameters assessed decline slowly from 0 to 48 h, with a sharp decline (p < 0.05) at 72 h of preservation. In conclusion, UHT and INRA-96 (R) were similar as base extenders, and the addition of 5% egg yolk plus 2% glycerol or just 5% egg yolk improved the quality of ram semen preserved at 5 degrees C, at least for 48 h. The combination of egg yolk-glycerol might provide extra protection in case of fluctuation of temperatures below 5 degrees C, commonly seen under field conditions.