The chemical composition of freezing extenders plays a major role in sperm cell survival during cryopreservation. In this study we compared two extenders for freezing stallion semen: INRA82 extender (as a control) versus INRA96 (R) extender, both supplemented with egg yolk and glycerol. INRA82 contains milk, whereas INRA96 (R) is a chemically- defined extender developed for fresh semen storage at 4 degrees C or 15 degrees C. Semen from 3 stallions (7 ejaculates per stallion) was frozen in both extenders. In vitro analyses of post-thaw motility of sperm cells (computer-assisted analysis) and of membrane integrity (flow cytometry analysis) were performed. Then a fertility trial was conducted. Inseminations were conducted in a total of 84 mare cycles. INRA96 (R) extender supplemented with egg yolk and glycerol significantly improved per-cycle pregnancy rates compared with INRA82 (71% versus 40%, p < 0.01). In agreement with these fertility results, membrane integrity was better preserved in INRA96 (R) than in INRA82. In contrast, motility parameters were significantly higher in INRA82 than in INRA96 (R). Further research is needed to understand how INRA96 (R) components protect sperm cells during the cryopreservation process and highly increase their fertility potential compared with INRA82 components.