The aim of the current study was to evaluate the effects of different concentrations of rainbow trout seminal plasma (RTSP) (0.1%, 1% and 10%) in extenders containing either egg yolk or lecithin for use in Awassi ram semen cryopreservation. Pooled sperm were diluted in a two-step dilution method to a final concentration of 1/5 (semen/extender) in egg yolk or lecithin extender containing no RTSP, 0.1%, 1% or 10% RTSP (v/v). Semen samples were assessed for sperm motility, plasma membrane integrity [hypoosmotic swelling test (HOST) and Hoechst 33258] and defective acrosomes [FITC-conjugated Pisum sativum agglutinin (PSA-FITC)] at the following five time points: after dilution with extender A; after equilibration; and post-thaw at 0 h, 3 h and 5 h. Malondialdehyde (MDA) was examined only after thawing. Freezing and thawing procedures (dilution, equilibration and post-thaw incubation at Oh, 3 h and 5 h) negatively affected the motility (P<0.001) and acrosome integrity (P<0.001). Additionally, freezing and thawing negatively affected the plasma membrane integrity, as determined by the HOST and Hoechst 33258 (P<0.001). The extender group affected the motility (P<0.001) and the HOST results (P<0.001). Levels of MDA in the egg yolk extender with 1% RTSP group were significantly lower than in the lecithin control group (P<0.05). In conclusion, the egg yolk extender groups that were supplemented with 10% and 1% RTSP provided greater cryoprotective effects for semen survivability during 5 h incubation than the other extender groups. (C) 2015 Elsevier B.V. All rights reserved.