This study was designed to compare Cortalap ® extender with tris-citric acid-glucose (TCG), Lepus ® and Merk III ® , to assess the in vitro preservability of rabbit spermatozoa stored for 72 h at 5°C. The best extender identified in vitro was then compared with fresh semen to determine fertility and prolificacy rates in vivo. Eight semen pools were split into four subsamples and each of them was diluted to a ratio of 1:10 (volume:volume) with four different extenders (Lepus ® , Cortalap ® , TCG and Merk III ® ) and stored at 5°C for 72 h. Sperm motility, viability, sperm membrane and acrosome integrity were evaluated in both fresh and chilled semen at 4, 24, 48 and 72 h of storage. The results showed that Cortalap ® was the best extender to preserve the quality of rabbit semen in vitro over 72 h in comparison with the other extenders (P<0.05). Then, Cortalap ® was used for an artificial insemination (AI) trial to be compared with fresh semen. Two groups of does (n=30 each) were inseminated with fresh and chilled semen. The fertility and prolificacy rates for the does inseminated with chilled semen were significantly lower compared with those inseminated with fresh semen (P<0.05). In conclusion, although Cortalap ® proved to be the best extender to preserve semen quality in vitro after 72 h at 5°C, the in vivo results showed that its use is not recommended in AI programmes.