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This study was designed to compare Cortalap^® extender with tris-citric acid-glucose (TCG), Lepus^® and Merk III^®, to assess the <em>in vitro</em> preservability of rabbit spermatozoa stored for 72 h at 5°C. The best extender identified <em>in vitro</em> was then compared with fresh semen to determine fertility and prolificacy rates <em>in vivo</em>. 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 <em>in vitro</em> over 72 h in comparison with the other extenders (P&lt;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&lt;0.05). In conclusion, although Cortalap^® proved to be the best extender to preserve semen quality <em>in vitro</em> after 72 h at 5°C, the <em>in vivo</em> results showed that its use is not recommended in AI programmes.